Poster Session

Malaria is a highly prevalent parasitic disease in regions with tropical and subtropical climate in the world. Developing a malaria vaccine is one of the greatest challenges in biomedical sciences. Among the species of Plasmodium-causing human malaria, P. vivax is the second most prevalent and the most geographically widespread species. In previous work, we generated two recombinant proteins representing the three allelic variants of P. vivax Circumsporozoite Protein (PvCSP) fused to the Mumps virus’s nucleocapsid protein, as a strategy to elicit strong immune responses. The objective of the present study was to evaluate the protective efficacy in mice of vaccine formulations containing Poly (I:C) and Montanide ISA720 as adjuvants. For this, we evaluated the performance of a new transgenic P. berghei parasite carrying the third PvCSP allelic variant, P. vivax-like, as murine challenge model. We also used our established infection model based on transgenic-CSP P. berghei/ P. vivax (VK210 and VK247) sporozoites. Formulations containing Montanide ISA720 were significantly more immunogenic and protective against experimental challenge of immunized mice than those adjuvated with Poly (I:C). Our results provide insights about the development of universal vaccines against malaria vivax.

Within the framework of the Covid-19 pandemic, caused by Sars-Cov-2, passive immunization is being considered as a clinical tool to alleviate and improve the treatment of people infected by the virus. The addition of antibodies from recovered patients has been shown to be short-term protection. Passive immunization with specific antibodies from other classes, such as egg yolks from hyperimmunized hens, enriched in Ig Y, could be another option since they are harmless.

We used 10 healthy White Leghorn hens from FARVET SPF (Ica, Peru) that were immunized at two-week intervals, with the supernatant purified from cell cultures expressing the recombinant Binding Protein Receptor protein (RBD) from Sars-Cov-2. The birds' serum was collected and tested by ELISA for anti-RBD antibodies and, they were purified from egg yolks using a novel methodology including biocompatible reagents such as vinegar and NaCl. These were tested in a double immunodiffusion assay and a Sars-Cov-2 neutralization (GenScript, USA) and proved positive. With these samples, we inoculated three-year-old pigs with 10 mg subcutaneously, and the serum was collected at 8 hours post-inoculation.  Then, it was used in neutralization tests with the active Sars-Cov-2 virus. The results showed a percentage of 100% inhibition compared to the positive controls. 

If these preliminary findings are corroborated, passive immunity with IgY could be another means of relieving patients infected with the virus and controlling the Sars-Cov-2 pandemic.

Plasmodium falciparum causes the severe form of malaria, responsible for over 400,000 deaths annually. Multiple lifecycle stages of the P. falciparum human malaria parasite are susceptible to antibody responses. Symptomatic malaria arises during the blood-stage, where merozoites of P. falciparum invade RBCs in a complex multistep process, which involves multiple interactions of parasite surface proteins and host receptors. Key to the parasite RBC invasion is the non-redundant interaction between reticulocyte-binding protein homologue 5 (PfRH5) and the RBC host receptor basigin (CD147). PfRH5 forms a complex with cysteine-rich protective antigen (PfCyRPA) and the PfRH5-interacting protein (PfRipr). The function of the PfRH5-PfCyRPA-PfRipr (RCR) protein complex is essential for invasion, as it has been described that conditionally disrupted genes for PfCyRPA or PfRipr abrogate merozoite invasion capability at the same point as when PfRH5 function is blocked. To date, several studies have demonstrated that induced antibodies by the antigens forming the RCR complex are able to block merozoite invasion and hence inhibit parasite growth. However, a significant challenge for vaccine immunologists is the identification of effective formulations of an antigen that can induce the high levels of antibody required to protect against complex parasites. We have established a novel ‘plug-and-display’ approach to enable the production of virus-like particles (VLPs) that can array antigens that have been refractory to genetic fusion. Here we present the development and production of VLPs displaying the antigens forming the RCR invasion complex as well as the immunological characterisation of their protective immunogenicity in vivo to small animals when administered in isolation, combinations and as the RCR complex. Our encouraging results demonstrate the clinical potential of this technology to develop vaccines able to elicit high levels of functional IgG that provide effective and sustained neutralisation of the blood-stage malaria parasite in humans. 

Cystic echinococcosis or cystic hydatid disease is a zoonotic parasitic disease with worldwide distribution caused by the larval stage of the Echinococcus granulosus. The development of an effective vaccine for the prevention and control of the disease is essential. EG95 protein of E. granulosus, is considered as a protective antigen and a good candidate in designing a beneficial vaccine against this parasite. Lactic acid bacteria (LAB) are non-pathogenic and non-invasive microorganisms. The surface display system of LAB such as Lactococcus lactis has provided a safe vehicle for antigen delivery and vaccine development. The eg95 encoding gene fragment fused to the M6 cell wall anchor was subcloned into the pNZ7021 expression vector and recombinant L. lactis NZ9000 displaying EG95 was constructed. The expression of an approximately 32-kDa EG95 protein was confirmed through SDS-PAGE, Western blotting, and immunofluorescence analysis. The immune responses were evaluated in BALB/c mice immunized orally and subcutaneously with live and killed EG95-expressing recombinant L. lactis, respectively. Total IgG level in mice immunized with heat-killed recombinant L. lactis (pNZ7021-eg95) was significantly increased compared to the control group. Mucosal sIgA was significantly higher in mice received live recombinant L. lactis (pNZ7021-eg95) compared to the control mice. Splenic lymphocytes from immunized mice represented high levels of IFN- γ, and low-levels of IL-4 and 10. Our findings suggest that immunization with EG95-expressing L. lactis induced both specific humoral and cellular immune responses in mice.

Recently, the sudden outbreak of devastating COVID-19 pandemic in December 2019 with its epicenter in Wuhan city of China, caused by Novel-CoV has toppled all the present medical system failing it badly to treat it. Still unstoppable and spreading at an alarming speed, with more than 2.5 million confirmed cases with a death toll over 8.5 lacs, undoubtedly with more than 18.5 million natural recoveries too. It has devastated all economy, shutting down all kinds of governmental and private businesses, making millions of people jobless. The vaccine or antiviral drugs are the most important treatments for COVID-19. The Peptide-conjugated morpholino-oligomers (PMO) and Tryphostin A9-RTKI are found in vivo and in vitro to have robust anti-viral activities with more than 80% efficacy in an antisense activity via p38 signaling against Coronaviridae, Mouse hepatitis virus (MHV), Porcine Epidemic Diarrhea Virus (PEDV) Transmissible Gastroenteritis Virus (TGEV), Porcine Epidemic Diarrhea Virus (PEDV) by stopping RNA replication. Our findings suggest that PMO-A9 combination decreases the viral replication with minimal toxicity demonstrated via p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK), indicating a possible treatment for COVID19 patients.

There is a need of adjuvants which can improve cellular immune responses of influenza vaccines. One interesting approach is to test different adjuvants in order to obtain enhanced Th1 immune response. Montanide 70VG is a water in oil adjuvant which has been used in numerous experimental veterinary vaccines. This adjuvant is good to induce humoral immune responses but little is known about the ability to induce cellular immune responses. In this work, we explored the properties of the Montanide 70VG, and in combination with the Poly(I:C) adjuvant, in order to enhance Th1 immunity of a universal influenza vaccine based on the recombinant nucleoprotein of influenza (rNP). We developed a vaccine formulated with rNP in combination with: the traditional incomplete Freund´s adjuvant (IFA), Montanide 70 VG, Poly (I:C), and Montanide 70 VG+Poly (I:C). 

The vaccine was administered in mice and humoral and cellular immune responses induced by the adjuvanted and non-adjuvanted vaccinations were evaluated. We found that combining Montanide 70 VG + Poly (I:C) adjuvants improved the Th1-skewed antigen-specific immune response. Vaccinated mice with the adjuvants combination developed the highest antibodies NP-specific titers of IgG. Interestingly, the use of Montanide 70 VG, in Montanide alone formulation as well in TLR3 combination, improved specifically IgG2b and IgG2c antibodies titers. In the contrast, the use of IFA adjuvant induced mainly IgG1 titer. In addition, vaccinated mice with Montanide 70 VG+Poly (I:C) showed a Th1–skewed response evidenced by a high frequency of IFN-γ-secreting CD4 T cells and also cytotoxic response evidenced by a high frequency of IFN-γ-secreting CD8 T cells by flow cytometry.  Our data demonstrated that rNP with Montanide 70VG plus TLR 3 agonist combination, is able induce mainly a Th1-immunity which is highly in demand to induce efficient immune responses against influenza. 

Plasmodium vivax is the most common species of human malaria parasite found outside Africa, with high endemicity in Asia, Central and South America, and Oceania. Although Plasmodium falciparum causes the majority of deaths, P. vivax can lead to severe malaria and result in significant morbidity and mortality. The development of a protective vaccine will be a major step toward malaria elimination. Recently, a formulation containing the three allelic variants of the P. vivax circumsporozoite protein (PvCSP-All epitopes) showed partial protection in mice after a challenge with the hybrid Plasmodium berghei (Pb) sporozoite, in which the PbCSP central repeats were replaced by the VK210 PvCSP repeats (Pb/Pv sporozoite). In the present study, the chimeric PvCSP allelic variants (VK210, VK247 and P. vivax-like) were fused with the mumps virus nucleocapsid protein in the absence (NLP-CSP_R) or presence of the conserved C-terminal (CT) domain of PvCSP (NLP-CSP_CT). To elicit stronger humoral and cellular responses, Pichia pastoris yeast was used to assemble them as nucleocapsid-like particles (NLPs). Mice were immunized with each recombinant protein adjuvanted with Poly (I:C) and presented a high frequency of antigen-specific antibody-secreting cells (ASCs) on days 5 and 30, respectively, in the spleen and bone marrow. Also, high IgG titers against all PvCSP variants were detected in the sera. Later, these immunized mice with NLP-CSP_CT were challenged with Pb/Pv sporozoites. Sterile protection was observed in 30% of the challenged mice. Therefore, this vaccine formulation use has the potential to be a good candidate for the development of a universal vaccine against P. vivax malaria. 

 Financing: FAPESP 2012/13032-5, CNPq-INCTV, CAPES.

A successful vaccine contains the protective antigen and an adjuvant that triggers innate and adaptive immune activation. The adjuvant is needed to intensify and guide the type of immune response generated. The lack of appropriate adjuvants may hinder the development of effective vaccines. The current challenge is to remedy the lack of adjuvants that drive efficient Th1 response in newborns, which is paramount to fight intracellular pathogens. We describe a novel function of a recombinant protein of Trypanosoma cruzi, TcX, which presents an adjuvant-like innate immune potential.

We showed that TcX induced the release of IFN-γ by NK cells and TNF-α by monocytes, NK and myeloid dendritic cells (mDCs). These cytokine responses were prominent in both cord and adult blood cells. Neutralization of the TNF-α response by specific antibodies reduced TcX-induced IFN-γ, indicating that TNF-α contribute to IFN-γ production by NK cells. TcX also upregulated co-stimulatory molecules on mDCs.

Otherwise, the use of HEK-TLR2, HEK-TLR4 and neutralizing antibodies allowed us to identify TLR2/1 and TLR4 as TcX receptors. NF-κB, MyD88 but not TRIF inhibitors reduced the TNF-α response to TcX. Finally, we tested the adjuvanticity of TcX on neonatal mice immunized against OVA. The TcX-adjuvanted group showed increased total IgG and particularly IgG2a antibodies to OVA, as compared to the alum-adjuvanted group.

Taken together our results suggest that TcX may function as a potent Th1-type pediatric vaccine adjuvant by promoting DCs, NK cells and monocytes activation and the release of IFN-γ and TNF-α downstream the TLR2/1-TLR4-MyD88 pathway.

Flagellin is a protein-based adjuvant and has been actively explored as vaccine carriers. Preclinical and clinical studies find flagellin-based vaccines have a risk to induce systemic adverse reactions potentially due to its overt activation of TLR5. To improve safety and immunogenicity of flagellin, FljB was displayed at high densities on hepatitis b core (HBc) virus-like particle (VLP) surface. FljB-HBc (FH) VLPs showed significantly reduced ability to activate TLR5 or induce systemic interleukin-6 release as compared to FljB. FH VLPs also failed to significantly increase rectal temperature of mice, while FljB could significantly increase rectal temperature of mice. These data indicated systemic safety of FljB could be significantly improved by high-density display on HBc VLP surface. Besides improved safety, FH VLPs and FljB similarly boosted co-administered ovalbumin immunization and FH VLPs were found to induce two-fold higher anti-FljB antibody titer than FljB. These data indicated preserved adjuvant potency and improved immunogenicity after high-density display of FljB on HBc VLP surface. Consistent with the high immunogenicity, FH VLPs were found to be more efficiently taken up by bone marrow-derived dendritic cells (BMDCs) and stimulate more potent DC maturation than FljB. Lastly, FH VLPs were found to be a more immunogenic carrier than FljB, HBc VLPs, or the widely used keyhole limpet hemocyanin for nicotine vaccine development with good local and systemic safety. Our data support FH VLPs to be a potentially safer and more immunogenic carrier than FljB for vaccine development.

Klebsiella pneumoniae is a Gram-negative bacterium of clinical importance, which colonizes mucosal surfaces and, from there, gains access to other tissues and establishes severe infections. Resistance to several antibiotic classes has been reported, making K. pneumoniae an increasing public health concern. Using a reverse vaccinology approach, we matched 8 publicly available genomes of K. pneumoniae and identified 528 conserved genes. To ensure they were conserved intraspecies, these genes have none to low homology to Escherichia coli K12 strain, an Enterobacteriaceae species like K. pneumoniae. Based on cellular location and size, 7 potential antigens were chosen, Klebs#3 to Klebs#9. Interestingly, Klebs#8 corresponded to the fimbrial protein MrkD, which has been extensively studied by others and is immunogenic. We next addressed their presence in 606 K. pneumoniae clinical isolates from Italian health structures (Careggi, in Florence, Tuscany and ISMETT, in Palermo, Sicily) and observed that Klebs#3, Klebs#7 and Klebs#9 were presented in more than 96.44%, 99.55% and 95.55%, respectively. Due to their high prevalence in K. pneumoniae clinical isolates, we believe that these 3 novel antigens should be further studied as components of a future immunogenic preparation against this pathogenic bacterium.

Background: Multi-drug resistant (MDR), especially extremely drug resistant (XDR), Mycobacterium tuberculosis (M. TB) is a big problem in the world.  We have developed novel TB therapeutic vaccine (HVJ-E/HSP65DNA+IL-12DNA vaccine) to eliminate MDR-TB by T cells.

Design/Methods: DNA vaccine expressing M.TB heat shock protein 65 and IL-12 was delivered by the hemagglutinating virus of Japan (HVJ)-envelope.  Human patient with MDR-TB was treated three times i.m. with 909μg pDNA + 909mNAU HVJ-E.

Results: This vaccine provided remarkable therapeutic efficacy against MDR-TB and XDR-TB in murine models (decrease in the number of MDR-TB).  Furthermore, this vaccine provided therapeutic efficacy of prolongation of survival time (100% survival) of TB infected cynomolgus monkeys (the best animal model) and augmented the immune responses.

【Preclinical study】

By using monkeys in GLP level, toxicological study of the vaccine showed little toxicity. Safety pharmacological study showed safety for central nervous, cardiovascular and respiratory systems.  

【Phase1 Investigator-initiated Clinical Trial】

Therefore, phase 1 clinical trial has been already started. Targets are human patients with MDR-TB. Primary evaluation is safety•tolerability.  Secondary evaluation is anti-TB efficacy (sputum-culture negative conversion).  A patient of First Patient In showed safety and tolerability of this therapeutic vaccine by pDNA concentration in the blood.  Furthermore, anti-TB efficacy was demonstrated by the Gaffky study and colony count of TB in the sputum.  Anti-TB immunity (IFN-γ and IL-2 production) was augmented in the patient with this vaccination from 14~126 day.

Discussion:  Safety and tolerability of this vaccine was shown in Phase 1 Clinical Trial.  Anti-TB efficacy and anti-TB T cell-immunity were demonstrated in vaccinated patient.  These data indicate that the vaccine is useful against MDR-TB through T cells for human therapeutic clinical applications.

Funding

This research was supported by the Research Program on Emerging and Re-emerging Infectious Diseases from Japan Agency for Medical Research and Development, AMED.

The Zika virus (ZIKV) is a single-strand positive RNA that belongs to the genus Flavivirus in the family Flaviviridae. The ZIKV Envelope (zE) glycoprotein is composed of three ectodomains (DI, DII, and DIII) and is involved in various aspects of the viral cycle, mediating binding and membrane fusion. The availability of tests for the laboratory diagnosis of Zika infection is still very restricted, so there is a need to develop an affordable and more accurate test, reducing the risk of cross-reaction. The Neutralizing antibodies reacting with the EDIII ZIKV are generally specific for each virus and do not cross-neutralize other viruses^. We pretend to express and analyze the EDIII domain of ZIKV in drosophila S2 cells to develop a rapid immunofluorescence diagnostic test able to detect the infection of ZIKV in serum of human patients. The fragment encoding EDIII-ZIKV was cloned in the pAcV5-HisA-Hygro and pMtBip- HisA drosophila vectors, and the product was transformed and screened into DH5α. The vector obtained, pAc_ZIKVEDIII-Hy , were transfected into S2 cells and after the selection we established the S2Ac_ZIKVEDIII-Hy cell line. The cell line showed fluorescence and was marked in Dot blotting. 

Despite the worldwide importance of infection caused by SARS-CoV-2, there remains no scientifically-based pharmacotherapy for COVID-19. Immunosuppressive drugs such as methylprednisolone (MP), dexamethasone (DM), rituximab (Rb), tocilizumab (Tb) have been used; however, we are awaiting demonstrations of dose-effects and/or side effects, effects of broad-spectrum use, and efficacy data. To facilitate this process, we conducted interaction analyses of active binding sites of the SARS-CoV-2 virus and ACE2. We also conducted an in silica study to determine whether there were any significant interactions between the M^Pro protease complex encoded by SARS-CoV-2 and ACE2. In silica data revealed that DM interacts with the SARS-CoV-2/ACE2 complex, interrupting progression of the infection, with affinity energy for SARS-CoV-2/ACE2/DM of -7.9 kcal/mol. Between the M^Pro protease virus and DM, we detected van der Waals interactions between THR169, VAL171, GLY195, THR196 and ASP197, hydrogen bonding with ASN133 and ALA194, and alkyl bonding with ILE308. For ACE2, van der Waals interactions and DM were found at PHE529 and TRP530, and alkyl bonding with VAL79, ILE83 and VAL304. In the interaction of the SARS-CoV-2/ACE2/MP complex were evidenced affinity energy data of -8.8 kcal /mol. Van der Waals interactions occurred between MP and the M^Pro protease at THR169 and ALA193, whereas, for ACE2, there were van der Waals interactions with VAL304, hydrogen bonding with GLU300 and ASN527, and alkyl and pi-alkyl bonds with VAL79, ILE83, PHE529 and TRP530. Monoclonal antibody class (Rb) interacted with active sites of ACE2 receptor and M^Pro, revealing a low energy cluster score of -1255, and high affinity for the SARS-CoV-2/ACE2 complex. Molecule-protein interactions (SARS-CoV-2/ACE2 DM, SARS-CoV-2/ACE2/MP), as well as protein-protein interactions (SARS-CoV-2/ACE2/Rb) interfered with viral replication by competing among the receptors for human and viral protease. Blocking these interactions would reduce viral load and production of inflammatory cytokines, enabling recovery of infected patients.

The human papillomavirus (HPV) vaccine Gardasil targets both low-risk (non-cancer-causing) and high-risk (cancer causing) HPV types, while the vaccine Cervarix only targets high-risk types, yet competition between HPV types is not well understood. We develop a within-host mathematical model to examine the long-term outcomes of HPV vaccination. We divide cells into low-risk and high-risk types, and examine the effect of competition for target cells, including the possibility of coinfection. We derive theoretical eradication thresholds for vaccine efficacy and illustrate the outcome for the two existing vaccines. If no vaccination occurs, both viral types coexist, whereas either or both viral types can be eradicated if the vaccine is sufficiently efficacious.

The emergence of SARS-CoV-2 and its pandemic spread was recognized by WHO as worldwide threat to public health. Therefore, rapid and straightforward access to high-quality biological material is crucial to accelerate research focused on genomic variation of SARS-CoV-2 and viral invasion on host cells, thus leading to antiviral drugs discovery and vaccine development. It is clear that collection of biospecimens should be carried out in organized and strictly regulated manner for the best outcomes to reduce the risk of pre-analytic bias. Gathering, preparation, and storage of samples have to be performed by professionally organized biobanks dedicated to quality management and assurance. 

To meet the urgent need for high-quality biospecimens, we established SARS-CoV-2 Section in Biobank of Medical University of Bialystok (MUB). All activities of samples collection, transportation, processing, and storage are described and regulated in Standard Operating Procedures (SOPs) according to CDC and WHO guidelines. Every process is standardized and performed in accordance with SOPs for maintaining quality and reproducibility. MUB Biobank is an integral part of SARS-CoV-2 Diagnostic Laboratory, where every diagnostic sample is biobanked that guarantees proper biospecimens flow and reduces risk of potential delays in samples preparation and preservation. Samples stored in MUB Biobank includes: 1)lysates (SARS-CoV-2-positive: 402, >27 000 negative), 2)RNA isolates (SARS-CoV-2-positive: 402, >27 000 negative), 3)plasma collected from patients clinically suspected of COVID-19: 76 aliquots, including 20 samples from SARS-CoV-2-positive and 56 from SARS-CoV-2-negative patients. Plasma samples are collected twice from each patient – first aliquot on the day of admission to hospital and second aliquot on the day of discharge. 

Such prospective collection of biospecimens obtained and preserved under standardized conditions along with comprehensive clinical data provides valuable material for advanced molecular analyses to understand viral genetic variability, immune response in COVID-19, development of vaccines, and implementation of novel genetic tests.

The ongoing COVID-19 pandemic has affected millions of people worldwide. The number of people infected continues to rise, while no antiviral therapy specific for COVID-19 is currently available. With multiple vaccines and drug candidates now in clinical trials, it is imperative to look for different strategies to combat the viral infection. Multiple viral protein targets involved in different mechanisms have been identified for therapeutics. Spike glycoprotein interacts with human ACE2 receptor to facilitate internalisation of the virus and is a popular choice for developing antibodies against SARS-CoV-2. N-linked glycosylation of the transmembrane spike protein is associated with viral shielding from antibody neutralisation, thus immune evasion. Masking of immunogenic epitopes due to glycosylation has also been studied for other coronaviruses. Recent mass-spectrometry studies have elucidated site specific signature of N-glycans on SARS-CoV-2 spike glycoprotein. This can also have implications in vaccine design. Carbohydrate binding agents (CBA) can target glycans of the glycoprotein and have an antiviral therapeutic potential. CBAs can range from proteins like, lectins to non-peptidic small molecules like; pradimicin A (PRMA). They have been shown to have anti-viral potential against a number of viruses like, human immunodeficiency virus (HIV), human hepatitis C virus (HCV), coronavirus and influenza virus. Lectins, Griffithsin and Urtica dioica agglutinin (UDA) have also been reported to have anti-viral potential in case of SARS-CoV glycosylated spike protein. Thus, studies involving in silico docking of lectins and SARS-CoV-2 spike glycoprotein can further reveal the interaction between the two proteins. Lectins can also be engineered by screening mutations that can increase the binding affinity for spike glycoprotein. Thus, this can open new avenues for SARS-CoV-2 therapeutics, that currently poses a serious threat to global health and economy.

Introduction

Developing a vaccine against SARS-CoV-2 is one of the most time-pressured scientific challenges of our time. Working at unprecedented speed, more than 150 vaccine projects are in development and 30 are in clinical phases. In this research piece, some of the most pressing issues in development have been considered. These include: (i) analysing viral mutability and its impact, (ii) collecting good-quality efficacy data in vaccine clinical trials, (iii) developing the manufacturing capabilities needed to satisfy demand, (iv) devising a global manufacturing and funding strategy. 

Methods

Scientific literature and experts’ opinion pieces were reviewed, and findings summarised to present the state of the art of vaccine development. An analysis of the evidence of viral mutations and their effect on vaccine efficacy was conducted.  Vaccine trial design was considered in the light of lockdown and social distancing measures. Vaccine manufacturing was discussed through an analysis of manufacturing platform technologies’ reports and COVID-19 funding provisions. A projection model was devised to present a future landscape of manufacturing capabilities. Lastly, a platform approach was proposed to help coordinate manufacturing and funding efforts. 

Results and Conclusions

While the antigenic profile of SARS-CoV-2 has not experienced obvious changes that are known to impact vaccine efficacy, a mutability monitoring system, combining sequencing and structural biology, should be adopted. Vaccine trial design can leverage previously acquired knowledge on correlates of immunity for coronavirus infections (MERS and SARS) and recent evidence from socio-behavioural and epidemiological studies to define efficacy assessment strategies. On the manufacturing front, the projection model shows that herd immunity could be reached by the end of 2022. The unprecedented manufacturing and funding effort leaves space for a new paradigm based on a global multi-tasking platform for manufacturing and licensing deals to ensure greater coordination.

Immunotherapy is the potential cancer treatment through the immune system response to eliminate the malignancies. The principle behind this method is to discover the specific target, that tumor-associated antigen is generally used. Cancer-Testis antigens are one of the tumor-associated antigens which can be a target of the immunotherapies. Among these, the MAGE-A family including 12 members from A1 to A12, has the highest immunogenicity. Several studies have suggested that among different tumor antigens the MAGE-A family can provoke the humoral and normal cellular immune responses in multiple malignancies including melanoma, lung, prostate, bladder, liver, and breast cancer. Hence, the MAGE-A family can be an acceptable candidate for immunotherapy.

In this study, a preliminary library of B-cell continuous epitopes from the MAGE-A family was predicted using the best immunoinformatics software including IEDB, IgPred, ElliPro, Bepipred, BCepred and, ABCpred. An overlapping-truncated library was designed based on the preliminary library of B-cell linear epitopes. Taken together, based on the truncated library, the best overlapping antigenic regions were identified among the majority of the MAGE-A family members, which can be an excellent candidate for antibody-based screening and cancer vaccine design.

We have modeled the kinetics of SARS-Cov-2 specific IgG, based on a single patient case (an Italian 57-year-old male, living in Pesaro (a province of Marche region being one of the main outbreaks of the epidemic in Italy), manifested the onset of symptoms (non-respiratory, but with diarrhea, asthenia, articular and muscle pain, anosmia, and ageusia), limited to the descending phase (during the ascending phase, the immunometric assays were not available yet). 

The blood samples were taken in the days 66, 95, and 133 from the beginning of the symptoms, and they were analyzed by CLIA chemiluminescence method, Megalumi® SNIBE.

Typical equations of first-order kinetics were used to describe the trend of the IgG concentration so that a negative exponential function was obtained: 

C = a e^-kt                        (1)

With some easy mathematical passages, from (1) we obtain

 ln(C) = -kt + ln a             (2)

The graph of the models is shown in the figure below. The results show an excellent determination coefficient (R² = 0.996). once we have the equation parameters, it is possible to calculate both the half-life of IgG (18 days) and the time of their negativization (170 days).

This result does not necessarily imply a loss of immunity at the expected time of negativization. In fact, as some authors have recently highlighted, a cell-mediated immunity could exist. However, our data may suggest the impossibility of identifying the subjects coming into contact with the virus for more than the time of negativization (in our case about six months since the onset of symptoms).

Despite the expected inter-subject variability, the perfect adaptation of the negative exponential model seems to be very promising and it suggests that it could fit equally well for other cases, with different parameters.   

Host cells have evolved a variety of sensors to detect infectious agents. These include the RIG-I-like receptors (RLRs) which recognize viral RNAs and the Toll-like receptors (TLRs) which have evolved to detect microbe specific molecules such as CpG DNA and lipopolysaccharides. These interactions trigger signaling events which activate key transcription factors such as interferon regulatory factor 3 (IRF3) and NF-κB, leading to the production of type I interferon (IFNs) and proinflammatory cytokines, which facilitate adaptive immunity. In addition there exists a key innate immune signaling pathway that has evolved to detect aberrant cytosolic DNA species, such as the genomes of bacteria or viruses following infection. This pathway is controlled by a cellular sensor referred to as STING (stimulator of interferon genes) that is activated by cyclic dinucleotides (CDN’s; cyclic di-GMP/AMP or cyclic GMP-AMP (cGAMP)) generated directly by invading bacteria, or via a DNA synthase cGAS (cGAMP synthase, also known as MB21D1) after association with viral or bacterial dsDNA. The STING pathway has been shown to be essential for stimulating host defense against an assortment of infectious agents. In addition, the extrinsic activation of STING in phagocytes via the DNA of engulfed dying cancer cells is essential for the generation of cytokines required for the efficient production of anti-tumor T cell responses. In contrast, chronic STING signaling has been shown to be associated with lethal autoinflammatory disease such as Aicardi-Goutieres Syndrome (AGS) and severe systemic lupus erythematosus (SLE). The activity of STING therefore requires tight control to prevent the sustained production of cytokines which are responsible for harmful autoimmune disease. Here, we will discuss the importance of STING-dependent innate immune signaling in controlling infectious disease as well as review the importance of STING agonists in vaccine design for the prevention of a wide variety of disease.

Background: Coronavirus disease 2019 (COVID-19) causes hyperinflammation, fibrosis, and tissue damage in the lungs, and several organs. An exponential rise in proinflammatory cytokines has been detected in patients with severe COVID-19 symptoms. Glycine (Gly), a nonessential amino acid in humans, plays a crucial role as an endogenous anti-inflammatory. Gly reduces proinflammatory mediators and their sensitivity to the cytokine signaling. SARS-CoV-2 mutates substituting aspartic acid with glycine in the spike (S) protein responsible for virus attachment and entry into the host. This confers virus’ virulence leading to the baseline deficiency in Gly content in COVID-19 patients, and consequently an insufficient response in curbing the inflammatory processes. We propose that Gly could be used both prophylactically and as an adjuvant treatment in COVID-19.

Methods: Databases used: Google Scholar, PubMed, and NCBI. Key search-descriptors: COVID-19, Gly in humans and SARS-CoV-2, Gly in COVID-19 treatment. Inclusion: Peer-reviewed sources fit the relevant search criteria, publications no later than 2005, PRISMA criteria for quality-check. Exclusion: Articles on other types of coronaviruses, pediatrics, animal models, pregnant women, editorials, and news articles. 

Results: A total of 22 articles were identified according to our search category, out of which 8 articles were selected based on the title, abstract, and purpose of our study. Research showed the role of Gly in humans and SARS-CoV-2, consequences of Gly deficiency, Gly in treating COVID-19. Gly inhibits the cytokine storm in humans. In mutated SARS-CoV-2, glycine replaces aspartic acid, increasing its virulence. Consequent baseline deficiency in Gly in the host, and a resultant increase in COVID-19 severity.

Conclusion: Dietary intake of glycine supplements as an adjuvant to the standard of care for COVID-19 patients may increase the survival benefits, mitigating cytokine storm, and tissue damage. Gly supplementation, orally or through nasogastric-tube, predominantly as an adjuvant in COVID-19 has not heretofore been investigated.

Purpose: Develop an epidemiological model capable of predicting intensity of COVID-19 cases for a potential site’s catchment population.

Methods: WAVE, a DICE dynamic transmission simulation of the COVID-19 epidemic, was constructed by fitting a hybrid model that considers an exponential growth phase (parameters: transmission rate, β; removal rate, γ) up to imposition of non-pharmaceutical interventions (NPIs); an exponential decay thereafter (parameters: Time constant rate, τ; maximum drop, κ); and potential regrowth (parameter: decay reversal μ) upon stopping NPIs. WAVE is calibrated to county-level new cases reported. Predictions were validated by comparison against subsequent case numbers. For each potential site, predictions are made for the period of likely recruitment and efficacy assessment based on its location-specific parameters. Scenarios consider reinstating NPI, reintroduction of cases from elsewhere and uncertainty in parameter estimates.

Results: One phase 3 SARS-COV-2 vaccine trial was planned after NPI were lifted in many US states but before recent surges emerged. Using WAVE, sites were selected for activation in areas predicted to see growing intensity during recruitment and subsequent weeks (e.g., in Phoenix, Arizona; Austin, Texas; Florida east coast, etc.). Surges have now been observed, validating those predictions. A second trial is selecting sites considering scenarios where NPIs are reinstated. To support site selection in other countries, local data are fit and resulting parameters fed to WAVE. Additional trials in planning phase are using WAVE.

Conclusions: It is possible to use DICE to simulate the epidemic trajectory at a granular enough level to inform site selection. Predictions from a hybrid model allowing alternating growth and decay phases as NPIs are imposed and inactivated are sufficiently accurate for this purpose. The model can also be used to inform COVID-19 treatment trials, trials for other vaccines, and non-COVID-19 trials that may, nevertheless, be affected by the pandemic.

The ALVAC-HIV clade B/AE and equivalent SIV-based/gp120 + Alum vaccines successfully decreased the risk of virus acquisition in humans and macaques. Here, we tested the efficacy of HIV clade B/C ALVAC/gp120 vaccine candidates + MF59 or different doses of Aluminum hydroxide (Alum) against SHIV-Cs of varying neutralization sensitivity in macaques. Low doses of Alum induced higher mucosal V2-specific IgA that increased the risk of Tier 2 SHIV-C acquisition. High Alum dosage, in contrast, elicited serum IgG to V2 that correlated with a decreased risk of Tier 1 SHIV-C acquisition. MF59 induced negligible mucosal antibodies to V2 and an inflammatory profile with blood C-reactive Protein (CRP) levels correlating with neutralizing antibody titers. MF59 decreased the risk of Tier 1 SHIV-C acquisition. The relationship between vaccine efficacy and the neutralization profile of the challenge virus appear to be linked to the different immunological spaces created by MF59 and Alum via CXCL10 and IL-1β, respectively.

Novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) is the cause of the deadly global coronavirus disease 2019 (COVID-19). This pandemic has become one of the most rapid and significant contributors to the global mortality burden. Immune evasion and rapid cell infectivity contribute to the fatal potential of this virus. Cell entry mechanisms of SARS-CoV-2 are crucial to understanding viral pathogenicity as well as for devising effective therapeutic intervention. Recent studies have elucidated the increased dependence of the buried SARS-CoV-2 S-protein receptor-binding domain (RBD) on pre-activation by human proprotein convertase, furin, reducing its dependence on target cell proteases, for effective exposure and binding to its receptor, human Angiotensin Converting Enzyme-2 (hACE2). This differentiates SARS-CoV-2 from SARS-CoV in maintaining efficient cell entry while remaining evasive to immune surveillance, thereby making furin a promising avenue for therapeutic intervention against SARS-CoV-2. Our study proposes a bispecific antibody for interrupting SARS-CoV-2 cell entry into human bronchial tissues through simultaneous blockade of SARS-CoV-2 S-protein and human furin, thereby presenting a novel therapeutic strategy against COVID-19. We present computationally affinity-matured sequences of camelid antibody Nb14 against human furin. The furin-inhibiting camelid antibody Nb14 VHH domain was subjected to in silico molecular docking studies, site-directed mutagenesis, mutant stability assessment and affinity maturation prediction of furin-mutant Nb14 complexes as part of our computational antibody affinity maturation protocol, resulting in 14 leads with considerable increased binding affinity to furin. We hypothesise that a bispecific antibody possessing two different affinity matured variable domain specificities against SARS-CoV-2 S-protein and human furin as resulting from our computational protocol, is capable of blocking invasion of human bronchial tissue by SARS-CoV-2 and holds promise as a potential treatment for COVID-19 after in vitro and in vivo validation.

Background: Coronavirus disease 2019 (COVID-19), declared a pandemic by the World Health Organization on March 11, 2020, has caused interruptions in routine pediatric care. Various lock-down strategies have further decreased the accessibility of routine immunization. The aim of this review is twofold (i) to evaluate the change in immunization coverage during the pandemic, and (ii) suggest strategies to improve pediatric vaccination rate.

Methods: A systematic review regarding the impact of COVID-19 on routine pediatric vaccinations was conducted. Literature search was performed using Embase, Scopus, Medline, Pubmed, Google Scholar, Web of Science, and CINAHL. A comprehensive search was completed using keywords 'Routine vaccination', 'Pediatric vaccination', 'Immunization’, and ‘COVID-19'. 

Results: Five studies were included that detailed decreases in vaccination rates, orders, and availability. Bramer et al. shows a decline in vaccination rates for all milestone age groups. Children up to 5 months of age had a reduction in annual vaccination from 67.9% in 2019 to 49.7% in May 2020. Mcdonald et al. showed a decrease in the hexavalent vaccine by 6.7% and MMR vaccines by 19.8% in 2020 compared to 2019. The author noticed not only a decrease in administration but also availability and demand. Vogt et al. noted 1,397 (81.1%) clinics offered immunization services, but <60% will provide immunization by August. Santoli et al. reported a reduction in orders for federally-funded organizations for non-influenza childhood and measles-containing vaccines. However, vaccine administration increased in late March 2020 for children ≤24 months. Chandir et al. in Pakistan noted daily pediatrics visits significantly declined by 52.8%. 

Although the scientific evidence is not  strong enough, it is believed that face masks may effectively prevent respiratory virus transmission from human-to-human. This study was undertaken to assess the use of facial masks among young adults during the COVID-19 pandemic in Poland. 

The survey was created with Google^Ò Forms and posted on Facebook^Ò groups for young people. The recall period was the last 7 days. At the time of data collection (12^th-14^th of April 2020) wearing face masks was not mandatory in Poland. Statistical analysis was performed with Statistica 13; Statsoft, Tulsa, OK, USA).

1393 responders (60.4%) declared use of face masks. There was no difference (p=0.12) in the frequency of face protection use between females and males. Interestingly, significantly more responders (p=0.007) who reported an atopic predisposition wore face masks (65.5% and 57.7%, respectively). Having self-reported sensitive skin did not influence the frequency of face masks wearing (p=0.06). 34.0% participants (significantly more males [p=0.0012]) used several types of face masks. Out of all usages, cloth masks (46.2%) appeared to be most popular ones, followed by surgical masks (39.2%), respirators (N95 and FFP) (13.3%), half-face elastomeric respirators (0.8%) and full-face respirators (0.4%). Females significantly more frequently (p=0.0001) used cloth masks. Concerning all the masks used, single-use masks constituted 52.5%. It appeared that 23.9% of responders who used single-use staff wore it again. 73.6% participants (significantly more females [p=0.004]) declared masks decontamination. Washing combined with ironing was the most common modality applied (63%), followed by the use of disinfectants (19.4%), ironing alone (4.8%) and putting into the oven (3.5%). 

Our results clearly showed that at least some practices among young people are inappropriate, which can reduce the effectiveness of face masks protection and allow the spread of infection. 

Background: Routine childhood immunization coverage in Pakistan remains sub-par, in part, due to the poor quality of service delivery. This study aims to summarize the quality of immunization centers throughout the Sindh province, Pakistan, by assessing all Government vaccination facilities and their associated health workers.

Methods: A health facility and worker assessment survey adapted from tools developed by BASICS and EPI-Sindh was used to record facility infrastructure, processes, and human resources. Using expert panel ranking, we developed critical criteria to indicate items immunization centers need to be operational (vaccinator, a cold box or refrigerator, and vaccine supplies) as well as high, low, and moderate function requirements and their respective scores. 

Results: Out of 1396 centers assessed, 1236 (88.5%) were operational, while 1209 (86.6%) offered immunization services. Only 793 (66.0%) of the functional immunization facilities met the critical criteria. The average scores for high, moderate, and low function requirements were significantly lower for centers that did not meet the critical criteria. Of 2,153 healthcare workers interviewed, 1874 (87.1%) were vaccinators. 1805 (96.3%), 1655 (88.3%), and 1387 (74.0%) were trained in vaccination, cold chain, and inventory, respectively.

Conclusion:  One out of three immunization centers in Sindh lack the critical components essential for quality vaccination services. Health workers have adequate training and experience, suggesting that sub-optimal vaccination center quality is due to inadequate infrastructure and inefficient processes. Our study presents innovative and critical research findings with high-impact policy implications for identifying and addressing gaps to improve vaccination uptake within an LMIC setting.

Background

The Covid-19 pandemic has enormously challenged healthcare systems globally. Exploring the lived experiences of nurses at the forefront of the fight against Covid-19 can help identify pivotal aspects and strategies, and provide important lessons to strengthen outbreak management and control for future epidemics.

Objectives

To examine 1) challenges encountered in combatting the Covid-19 pandemic, and 2) strategies to prepare for future infections.

Methods

Semi-structured individual interviews were conducted with 39 registered nurses who have provided care for patients diagnosed with Covid-19 or with suspected Covid-19 symptoms. Nurses were recruited from acute care settings, including medical wards, intensive care units, isolation wards, and emergency departments, in Hong Kong. Participants were asked to share personal, community and global challenges encountered in combatting Covid-19, the strategies adopted to address them, and effective measures for future epidemics. All interviews were audio recorded and transcribed verbatim for thematic analysis.  

Results

Participants identified the high transmissibility of the Covid-19 virus, global personal protective equipment shortages, insufficient community health education, and lack of an effective treatment or vaccine as the major challenges in fighting against the Covid-19 pandemic. Majority of participants emphasised the need for a long-term solution against the disease in place of the current approach of only attempting to relieve patient symptoms. Multiple strategies in preparation for future epidemics were suggested, including more comprehensive and ongoing nursing education on outbreak management, increased number of isolation facilities, public education on hand hygiene and mask-wearing, faster response mechanisms of hospitals, governments and organisations, and early treatment of patients in the absence of vaccines.

Conclusions

While effective vaccines and treatments are required to offer more permanent solutions to infectious diseases, they take long periods of time to be achieved. Consequently, we must meanwhile rely on other proposed strategies focusing on earlier and faster plans of action.

Background:   

Coronavirus’s evolution is presumably a natural recombination of RNA viruses, but deterioration and remission in clinical manifestations remain diverse. SARS-CoV-2 might coexist with human carriers through continuous recombination. COVID-19 has been identified to create multiple genomic variations, the potential basis for susceptibility. The significant mutations that increase transmissibility are S protein, nucleoprotein, RNA polymerase, and RNA primase. Genomic alterations allow faster replication in SARS-CoV-2 and are unique for two different RNA-dependent RNA polymerases; one is a primer dependent protein; another is Nsp8, having autonomous primase capacity. The cell entry receptors, ACE2 polymorphisms, may dictate SARS-CoV-2 susceptibility. Four regulatory SNPs from TMPRSS2 and one from CD26 have a significant role in regulating SARS-CoV-2 infection genes. 

Methods: 

A systematic literature review from 8/14/20-8/24/20, targeting data from the date of origin of COVID-19. We included age 18 years or older and studies focusing on genotypes and excluded children and articles in different languages. We analyzed the studies on genotypic variations in Coronavirus and humans. 

Results:

From a total of 25 articles, 16 met the inclusion criteria. We identified the critical factors involved in COVID-19, including S (spike), N (nucleoprotein), and E (envelope) proteins. S protein's role in receptor-binding and membrane fusion indicates that vaccines based on the spike protein could induce antibodies to block virus binding and fusion or neutralize infection. Differences in the spike's length may play an essential role in the pathogenesis and treatment. Nsp8 based vaccines reduce virulence and generate T-cell responses. Therefore, high-frequency SNP mutation sites must be considered when designing a SARS-CoV-2 vaccine. However,  missense variations from TMPRSS2 and CD26/DPP4 result in variations globally that need to be conceded. 

Conclusion:

These key factors in the host and viral genome are the possible target sites of potential vaccines and therapeutics for novel coronavirus. 

Background: The SARS-CoV-2 pandemic poses a significant global threat, with over 16 million confirmed cases and at least 650 000 deaths from COVID-19 in the first 7 months of the pandemic. Developing tools to measure seroprevalence and understand protective immunity to SARS-CoV-2 is a priority. We aimed to develop a serological assay using plant-derived recombinant viral proteins, which represent important tools in less-resourced settings.

 Methods: We established an indirect enzyme-linked immunosorbent assay (ELISA) using S1 and receptor-binding domain (RBD) portions of the spike protein from SARS-CoV-2, expressed in Nicotiana benthamiana. We measured antibody responses in sera from South African patients (n=77) who had tested positive by PCR for SARS-CoV-2. Samples were taken a median of six weeks after the diagnosis, and the majority of participants had mild and moderate COVID-19 disease. In addition, we tested the reactivity of pre-pandemic plasma (n=58) and compared the performance of our in-house ELISA with a commercial assay. 

 Results: SARS-CoV-2-specific immunoglobulins are readily detectable using recombinant plant-derived viral proteins, in patients who tested positive for SARS-CoV-2 by PCR. Reactivity to S1 and RBD was detected in 51 (66%) and 48 (62%) of participants. Notably, we detected 100% of samples identified as having S1-specific antibodies by a validated, high sensitivity commercial ELISA, and OD values were strongly and significantly correlated between the two assays. For the pre-pandemic plasma, 1/58 (1.7%) of samples were positive, indicating a high specificity for SARS-CoV-2 in our ELISA. SARS-CoV-2-specific IgG correlated significantly with IgA and IgM responses. Endpoint titers of S1- and RBD-specific immunoglobulins ranged from 1:50 to 1:3200. 

 Conclusions: We demonstrate that recombinant SARS-CoV-2 proteins produced in plants enable robust detection of SARS-CoV-2 humoral responses. This assay can be used for seroepidemiological studies and to measure the strength and durability of antibody responses to SARS-CoV-2 in infected patients in our setting.

With current preferment in computational biology, the arena of antibody modeling for efficacious interaction between active residues of antigen and antibody has grown at a humongous rate. Computer-aided affinity maturation mediates highly stable engagement between the active domain of paratopes (antibody) and epitope (antigen). Biomarker CD3 in complex with the T-cell receptor (TCR) activates T-cell. However, various studies have demonstrated that anti-CD3 antibodies alone are capable of T-cell activation. Anti-CD3 antibodies, alongside as monoclonal antibody, can also be used as a capable neutralizing arm in a bispecific antibody. In the present study, our paramount objective is in silico affinity maturation of the anti-CD3 paratope domain for optimum binding efficiency. Thus, facilitating specific interaction between the target and the therapeutic proteins (antibodies). Another objective of this research study is to screen out antibody-based therapeutic candidates possessing highly stable engagement with higher binding affinity among already existing antibodies. The sequential order of methodology involves retrieval of antibody sequences from open patent databases and literature followed by BLAST (Basic Local Alignment Tool) and MSA (Multiple Sequence Alignment) for evaluating the occurrence of the conserved and hypervariable domain. By homology-based protein structure prediction tools, antibody modeling with the subsequent screening of CDRs (Comlementarity Determining Regions) by Parapred is assessed. Further, for systematic evaluation of binding affinity between active residues taking part in the interaction, protein-protein interaction via in silico docking has been carried out for about fourteen lead candidates. Site-directed mutation, and selection has been performed, followed by data interpretation of ∆G (free energy) and various other parameters were evaluated before and after structural modifications to screen out the best interacting Ab-Ag complexes. This approach can be devised forward in the future for immunotherapy for assessing the lower energy Ag-Ab complex among the already existing ones.

Chlamydia trachomatis (Ct) is the most frequent sexually transmitted bacterial infection worldwide. Infections in women can lead to infertility, chronic pelvic pain and ectopic pregnancy. Nevertheless, a vaccine is currently not available partly due to the lack of appropriate animal models. The most critical and most expensive phase in vaccine development is phase III clinical trials. In this phase, an STD vaccine must demonstrate immunogenicity and efficacy in an at-risk population. This population is not only outbred, but to the vast majority also pre-exposed to the STD. Therefore, an animal model that can accurately predict the outcome of this phase must model this outbred pre-exposed population. Nevertheless, such an animal model has been missing. To overcome that limitation, we performed a proof-of-principle vaccination study using an intranasal administration of Chlamydia suis (Cs) as model pathogen with and without the TriAdj adjuvant. Cs is a close relative to Ct with demonstrated immunological cross-reactivity. Our results demonstrate that this pre-exposed outbred pig model can be used to demonstrate vaccine efficacy and immunogenicity: Our vaccination trial showed that compared to unvaccinated animals, pigs from both vaccinated groups had a reduced genital Cs burden. Furthermore, we could demonstrate that both vaccinated groups, but mainly the TriAdj adjuvanted group, induced IFN-γ producing CD4⁺ cells and their differentiation into tissue-draining effector memory cells. Thereby, we not only showed vaccine efficacy and immunogenicity in a highly translatable animal model, but we also validated the usefulness of the outbred pre-exposed pig model to predict the outcome of phase III clinical trials for Ct vaccines.

Background: The Integrated Management of Childhood Illnesses (IMCI) strategy was launched by WHO and its partners in 1995 to reduce child mortality by enhancing frontline health workers’ (FHWs) ability to diagnose and manage childhood illnesses, and strengthen overall health systems. However, although IMCI is associated with decreased child mortality, numerous studies have shown its impact has been limited due to persistent barriers to implementation.

Method: We developed a digitized version of IMCI (eIMCI) using Android technology to overcome implementation problems, including poor protocol compliance, lengthy trainings, paper-based data collection, and gaps in the referral system. The feasibility and efficacy of eIMCI was assessed through a mixed-methods pilot study encompassing baseline and end-line surveys, interviews, and Focus Group Discussions, in a low-resource rural district of Punjab, Pakistan. During the 8 weeks of deployment, a total of 1,978 children were enrolled in the eIMCI application by 10 participating FHWs, and 47 electronic referrals were generated. 

Results: Preliminary outcomes showed reduced disease occurrence (for example, diarrhea decreased from 48% at baseline to 29% at endline), increased adherence to IMCI protocol, and strengthened health system linkages (facility referrals increased from 5% at baseline to 45% at endline). FHWs reported enhanced performance, as well as an improved community response to their services.

Discussion: Indicating the feasibility and efficacy of eIMCI deployment in a low-resource setting, evidence from the pilot demonstrates the benefit of digitizing the IMCI protocol. eIMCI is a digital health solution with the potential to significantly reduce child mortality and improve service delivery and performance of FHWs.

Influenza, a highly contagious respiratory illness, remains a significant threat to public health. A broadly effective vaccine that could protect not only against seasonal influenza variants but also zoonotic and emerging pandemic influenza strains is urgently needed. Attempts to elicit such cross-protective immunity are mainly based on conserved protein targets such as long alpha-helix (LAH) from hemagglutinin stalk and ectodomain of matrix protein 2 ion channel (M2e). Recently, we reported the 3D structure and some practical applications of LAH expressed in the Escherichia coli system (referred to as tri-stalk protein). Here, we investigated the immunogenicity and efficacy of a panel of broadly protective influenza vaccine candidates based on influenza tri-stalk and triple M2e protein (3M2e) integrated into bacteriophage AP205 virus-like particle (VLP) platform, alone or in combination. While VLPs containing the 3M2e alone induced protection against standard homologous and heterologous virus challenge in mice, only the combination of both conserved influenza antigens into a single VLP fully protected mice from a high-dose homologous H1N1 influenza infection. We suggest that merging of genetic fusion and chemical coupling techniques to present multiple conserved influenza antigens on a single particle is a perspective approach for a broadly-effective vaccine candidate that could protect against the constantly emerging influenza virus strains.

The impending coronavirus disease 2019 (Covid-19), and the risk of its worldwide pandemic development requires urgent effective therapeutic procedures, being it orphan of a specific vaccine predictably for the next 24 months or more.

Attempts to treat the infection with some chemotherapies borrowed by other antiviral registered schedules or with other adopted off label drugs are on the way, but our concern is to run against the time adding if possible a further safe, effective sustainable treatment available to the greatest number of people anxiously claiming for prevention or disease control.

We focused on the challenge of an impressive bacterial quick and safe antiviral activity, incidentally observed on advanced metastatic cancer injected on a compassionate spontaneous basis with dead bacteria into the tumor masses (years 1975-79). 

Indeed, we had an experience namely with Corynebacterium parvum which displayed the best effective and safe viral symptoms remission in comparison with BCG and one autochthonous wild strain of Streptococcus faecium.

We successfully followed up with this procedure occasionally in the more recent years in some cases voluntarily requiring vaccination after detailed informed consent acceptance, to relieve their aggressive cumbersome viral infections.

The take home message of this abstract is to take in account not only the excellent new tailored Gene targeting therapies, but, especially in emergency, to do not absolutely forget the old ones!!!

In this study, the potential of N-trimethyl chitosan (TMC) nanoparticles as a carrier system for the nasal delivery of the r4M2e.HSP70c, as an M2e-based universal recombinant influenza virus vaccine candidate, was investigated in mice. The anti-M2e specific cellular and humoral immune responses were assessed and the protective efficacy against a 90% lethal dose (LD90) of influenza A/PR/8/34 (H1N1) in a mice model was evaluated. 

Our results showed that the intranasal immunization of mice with r4M2e.HSP70c+TMC rather than the control groups, r4M2e+TMC, r4M2e and PBS (Phosphate buffer saline), significantly elevated both longevity and serum level of the total M2e-specific IgG antibody with a significant shift in the IgG2a/IgG1 ratio toward IgG2a, induced a Th1 skewed humoral and cellular immune responses, increased IFN-y, IgG, and IgA in the bronchoalveolar lavage fluid (BALF), and promoted the proliferation of peripheral blood lymphocytes with lower morbidity and mortality rate against viral challenge. 

In conclusion, based on evidence to our finding, nasal vaccination with r4M2e.HSP70c antigen encapsulated into N-Trimethyl Chitosan (TMC) nanoparticulate system showed to induce a long lasting M2e-specific humoral and cellular immune responses and also provided full protection against a 90% lethal dose (LD90) of the influenza virus A/PR/8/34 (H1N1). It seems, protective immunity following intranasal administration of r4M2e could be resulted by the cooperation of both adjuvants, TMC and HSP70c.

Introduction: COVID-19caused by SARS-Cov-2 Virus as a novel virus newly emerged in humans.Till date there are no vaccines or treatments for covid-19right now. As the pandemic state still exists reverse vaccinology technique was used to to identify the potential vaccine candidate. The peptide-based synthetic vaccines have many advantages in terms of safety, efficacy and cross-protection, with reproducible, cost-effective and large-scale production, to fight COVID-19. 

Aim:1. to identify the pepetide which could be a potential candidate for the vaccine by insilico methods. ( reverse vacinology)

2.To study the safety of the vaccine in animals ( hamster)

Method: By using Insilco technique an epitope has been identified as a potential vaccine candidate against COVID-19 using Reverse Vaccinology (RV) approach. The safety testing in animals is carried out under guidelines mandated by Control and Supervision of Experiments on Animals (CPCSEA). These tests are conducted to evaluate vaccine safety in hamsters in two groups – one receiveing high dose and another low dose of vaccine for a period of 120 days with assesemnt done in 30 , 90 and 120 days. 

This will lead to assessment of the ability to raise antibodies to the virus and its levels in the system. The cytokine storm which the virus produces also can be assessed after the vaccine is given.

The safety of the candidate vaccine SPV and occurrence of serious adverse events (SAEs) evaluated will help to identify potential target organs of toxicity and to determine whether the toxicity is reversible after a period of time following the termination of treatment. Evaluation of immunogenicity and efficacy of the candidate 

This Synthetic Peptide vaccine (SPV) against COVID-19 will help  in identifying the ability of a vaccine to induce an immune response (antibody- and/or cell-mediated immunity) in a vaccinated  animals againstSARS-CoV-2 virus and then continue with the human trials.

Influenza pandemic and epidemic, high mutation frequency of the virus, and problems related to vaccine production capacity and cost necessitate the development of next-generation vaccine candidates that are not only broadly cross-protective, but also efficient in terms of cost and production time. One of the ways to explore the solution to this problem is to develop influenza Tc-epitope-based vaccines with the MPyV VLP as a carrier. In this research, MPyV VLP vaccines carrying influenza Tc-epitopes either externally or internally were developed. An MPyV VLP carrying Tc-epitopes at a surface-exposed loop of VP1 subunit protein was further developed by optimising assembly by co-assembling modified DDIT1 capsomeres with unmodified capsomeres to form stable mosaic VLPs. Furthermore, an MPyV VLP carrying Tc-epitopes internally was developed by encapsidating soluble subdomains of the M1 protein. To analyse and characterise the modified VLPs, a new semi-preparative SEC method was developed and validated with wt VLPs. Capsomeres bound to M1-I subdomains (VP1:VP2C-M1-I) were successfully expressed and purified as indicated by SDS-PAGE and western blot. VLPs of VP1:VP2C-M1-I and mosaic DDIT were also successfully assembled and purified with the newly developed semi-preparative SEC method. Analysis and characterisation of elution fractions with DLS, SDS-PAGE, and TEM showed successful formation of the desired VLPs, both DDIT1 mosaic VLPs and M1-I encapsidating VLPs.

Background: The four evolutionary stages of manufacturing have brought us to the emergence of Vaccine 4.0, a technology commonly referred to as Industry 4.0. Using the basic principle of connecting systems and machines, we posit new intelligent neural networks along the vaccine value chain control one another, predict failures, autonomously enable maintenance processes autonomously, self-organize logistics, and react to changes in production. However, the proposed framework requires a workup before prescribing this technology adoption.

Methods: Two challenges emerge before embarking on the path to Vaccine 4.0: first, the key technologies required for radical digital transformation in the manufacturing, production, rolling-out, and distribution process, and second, understanding before undertaking. Vaccine 4.0 analytics proposedly cater to 1) process performance, 2) process development, 3) process stability, 4) compliance, 5) quality assessment, and 6) optimized maintenance. The immediate benefits of digitization during the COVID-19 era are the continued assurance of process control, and the potential of big-data analytics to accurately detect deviations from set parameter limits. Big-data analytics may be utilized to improve the quality of vaccines, profitability, and the entire manufacturing process. The path to Vaccine 4.0 will enhance quality, improve efficacy, and compliance with data-regulated requirements (figure 1).

Results: Logistical and fiscal barriers are especially present in resource-limited settings within and across different countries. The proposed framework considers the potential barriers to manufacturing and equitable global distribution of COVID-19 vaccines. Use of big-data analytics and biometrics has potential to identify pockets of vulnerable populations who are at higher risk of transmission. Artificial intelligence enabled sensors and robotics support thermostable vaccine distribution in low capacity regions, while biosensors detect vaccinations that have low efficacy and vaccine blockchain systems support distribution in resource-limited settings.

Conclusion: Our proposed framework sets the path towards Vaccine 4.0 by creating an industrious and valuable new era of manufacturing.

Background: Despite free routine immunizations in LMICs, high default rates impede progress towards optimal immunization coverage, and without the technology to visualize the risk of large datasets, vaccinators and policymakers are unable to identify these potential defaulters. This study aimed to conduct feasibility testing and validation of a machine-learning-based predictive analytics algorithm that leverages artificial intelligence and uses statistical modeling and multidimensional data mining to accurately identify children who are most likely to delay or miss their follow-up immunization visits.

Methods: The algorithm was developed using 47,554 longitudinal immunization records, classified into the training and validation cohorts. Four different machine learning models were used to generate the predictive algorithm using a range of socio-demographic variables as predictors of defaulting. Each of the models was assessed in terms of accuracy, precision, sensitivity, specificity and area under the curve (AUC).

Results: Out of 11,889 cases in the validation dataset, the random forest model correctly predicted 8994 cases, yielding 94.9% sensitivity, and 54.9% specificity. The C-forest model, SVMs, and recursive partitioning models improved prediction by achieving 352, 376, and 389 correctly predicted cases, respectively, above the predictions made by the random forest model. All models had a C-statistic of 0.750 or above, whereas the highest statistic (AUC 0.791, 95% CI 0.784-0.798) was observed in the recursive partitioning algorithm.

Discussion: This feasibility study demonstrates predictive analytics can accurately identify children who are at a higher risk for defaulting on follow-up immunization visits. Correct identification of potential defaulters opens a window for evidence-based targeted interventions in resource-limited settings to achieve optimal immunization coverage and timeliness.

Background: Low immunization coverage rates have prompted the need to design effective demand-side interventions to improve uptake. Simple SMS reminders and large (≥USD 100) incentives have been demonstrated to effectively increase immunization coverage. However, research on small incentives (≤USD 15) for immunization is sparse and there is uncertainty regarding the optimal structure of incentives in terms of size, schedule, and design. 

Methods: A 12-arm randomized controlled trial is being conducted in Karachi, Pakistan with 10 incentive arms of varying size (amount), schedules (progressivity) and design (certainty of payment), an exclusive SMS arm and control arm. The incentives are provided through two mechanisms: mobile air time and mobile-money through a mobile-money provider. Children were enrolled when they presented to the study sites for either BCG, Penta-1, or Penta-2 vaccine.

Results: A total of 11,197 children were enrolled between Nov 2017 and Oct 2018 with approximately 800 children in each incentive arm and 1600 and 1599 in SMS and Control arm, respectively. As of Jan 09, 2020, Penta-3 and Measles-1 coverage are lowest in the control arm (71% and 60% respectively) and highest in the high-flat-lottery arm  (78% and 69% respectively) with uniformly progressing incentive amounts along with the immunization schedule.

Discussion: Interim results suggest that small mobile-based incentives coupled with SMS reminders can significantly improve immunization coverage in children. The final analysis will provide robust evidence of this impact and provide policy implications for the most optimal incentive structure that can build caregiver demand for effectively increasing child immunization in an LMIC setting.

Background: Despite the provision of free-of-cost vaccines in Pakistan, fully immunized child (FIC) coverage in Sindh province remains low at 49%. In 2012, we developed and piloted the Zindagi Mehfooz (Safe Life; ZM) Digital Immunization Registry, an Android-based platform that enables vaccinators to enroll and track child level immunization data of children in the catchment population. In 2017, ZM was scaled-up across Sindh province and is currently being used by 2,284 vaccinators across 1,526 facilities serving >48 million (m) population.

Method: All children under-2 years of age visiting EPI centers are enrolled. At enrollment, caregiver and child bio-data and child immunization history are recorded, and the child is provided with a unique Quick Response (QR) code for identification. For follow-up immunization visits, 3 SMS reminders are sent to caregivers, and upon immunization, child history is retrieved by scanning the QR code and vaccination record updated. ZM allows real-time access to data and generation of monitoring reports. Data from ZM was used to calculate coverage rates, timeliness, and trends for immunization coverage in Sindh.

Results: From Oct’17 to Dec’19, more than 2.4m children and 0.8m women were enrolled in the Registry, while >17m immunizations were administered. The FIC coverage in 12-23 months old children has increased from 49% (at baseline) to 57% for children enrolled in ZM. Additionally, pentavalent-3 coverage increased from 59% to 68%.

Discussion: ZM demonstrates the potential of DIRs to improve immunization outcomes within low-resource settings by enabling better child tracking and a higher retention rate. Additionally, the big dataset provides the opportunity to identify real-time trends and provides actionable data for evidence-based decision making.

Introduction 

In contrast to the global trends which have seen a rapid rise in the cases of measles, the Measles and Rubella (MR) campaign conducted in selected states is attributed to the reduction in measles and rubella cases 

Methods 

We conducted a mixed-method approach with in-person and virtual interview based on the structured questionnaire at OPD and through virtual telephonic interaction, respectively, to assess the parents’ knowledge and perception about the Government of Maharashtra, India MR campaign, driven through schools in a limited resource setting 

Results 

418 parents were surveyed. The mean age of children was 7.2 years (minimum 0.4, maximum 16, SD ± 3.8, 95% CI 6.9 to 7.6). 57.17 % (n=239) parents were undergraduate. 84.92 % (n=355) were of low-income group. 231 mothers (55.2%) were housewives. 71.5% (n=299) of the mother or father were decision-makers for the administration of MMR vaccine. The highest trust for the decision to administer vaccine was on local paediatrician (n=311, 74.4%). The fear of side effects was the primary reason to deny vaccine (n=231, 55.2%), 71.2 (n=298) believed that the campaign has been politically motivated. The availability of the vaccination centers all throughout 24 hours would have facilitated better uptake (n=292, 69.8%) and geographical proximity would have driven higher vaccination rates (n=331, 79.1%) 

Discussion

We identified information and knowledge gaps as the barriers to accepting MR campaign by the parents. We postulate that in the era of patient empowerment, it is important to deliver factual information for the better implementation of the campaign

Background 

HPV vaccine has been a revolutionary prophylactic anti-cancer vaccine against cervical cancer. The FDA approved the use of the vaccine in 2006 for all females aged 9-26 years and it was believed to extend protection to males through herd immunity.

Aim and Methods

The objective of this study was to do a literature review to assess the need for routine HPV vaccine in boys, and to analyze whether the surge in the number of oropharyngeal cancers and males having sex with males (MSM) warrants the need for more targeted HPV immunization program. 

Results 

Routine HPV vaccination in males was introduced in the US after ACIP recommended the use of HPV vaccine in boys of 11-12 years and a catch-up vaccination from 13-21 years, although in comparison to females the rate remains insufficient. Studies have also confirmed the comparable effectiveness of HPV vaccines between 16-26 years and 27-45 years in both males and females, but the expense of the vaccine remains a major challenge.

HPV vaccine when used routinely in males, was noted to reduce the number of total anal cancers by 75% in an Amsterdam study, and it was also found effective in MSM, even when administered after sexual debut, in reducing the risk of HPV as per the Québec Immunization Committee.

HPV can also cause oropharyngeal cancer, which has shot up over the past few years, especially among males in the age group of 40-60 years in developed countries, attributing to oral sex, further legitimizing the need for routine administration of HPV vaccine in males.

Conclusion

Gender-neutral immunisation against HPV needs to be implemented to protect both males and females of all the age groups against HPV not only to reduce the risk of HPV but also to reduce the stigma and inequality in HPV-driven disease in MSM. 

Background:

Pakistan has the third largest unvaccinated childhood population with a childhood immunization coverage of 66%, with significant inequalities in vaccine coverage between urban, rural, and slum areas, especially areas distantly located from vaccine centers. Frequent outbreaks of vaccine-preventable diseases such as polio and measles, in urban cities like Karachi, a result of pockets of un- or under-immunized children, signal the need for improved vaccine services in underserved areas. For this purpose, in April 2019, we introduced the Mobile Immunization Van (MIV) initiative in Karachi in collaboration with EPI Sindh. 

Methods:

Prior to each MIV visit, mobilization efforts are conducted in targeted areas to encourage caretakers to bring all children under 2 years of age for vaccination.  The MIV is then parked at a central location, and announcements made via loudspeakers to inform caregivers of the MIV and the importance of vaccinations. All vaccinations are administered in the MIV, and entries recorded in the Government’s Digital Immunization Registry (DIR) along with GIS coordinates. 

Results: 

Between April 2019 and January 2020, 2,867 children were vaccinated through MIV’s. Of those immunized through MIV’s, 50% were zero dose, i.e. having never been immunized previously, and 80% children late for their scheduled follow up vaccines. GIS analysis of MIV’s data confirmed that immunizations were conducted in slums, as well as areas distantly located from EPI centers. Moreover, compared to government outreach activities, 5% more children received BCG,  6% Penta3 and 4% Measles1 through MIV’s.

Conclusion:

Mobile Immunization Vans provide an opportunity to immunize zero dose children as well as vaccine defaulters from the most underserved hard to reach areas, while simultaneously enrolling them in the Government’s DIR allowing effective evaluation of immunization coverage.

Background: Despite progress in increasing immunization coverage, poor uptake of vaccines remains a challenge, particularly in LMICs. In part, this is due to poor socio-economic status of caregivers, lack of motivation, and parental procrastination. While there is evidence that large (≥100 USD) incentives can overcome these problems, research on small incentives that are more appropriate for an LMIC setting is sparse. Our study addresses this gap by evaluating the impact of small (≤12 USD) CCTs in increasing immunization coverage for children under the age of two.

Methods: A 2-year program utilizing two small incentive schemes of different amounts (high: USD9; low: USD5) was implemented in Shikarpur District, Pakistan. Caregivers (with children 0-24 months) visiting any of the 33 study immunization centers were given the opportunity to enroll.  All consenting parent-child pairs were randomized in one of the two incentive schemes and incentives were disbursed following each immunization visit, using mobile money.  A baseline and end-line survey was conducted to evaluate changes in pre-post coverage. 

Results: A total of 63,336 participants were enrolled between March 2016 - April 2018, of which 41,483 were randomized in the high or low incentive schemes (remaining caregivers were enrolled in the no-incentive group). Results show a 13% and 28% increase in age-appropriate coverage for Measles-1 (M1) and fully immunized children (FIC) compared to the baseline (FIC:16%, M1: 38%). There was no difference in M1 coverage between the high and low incentive schemes.

Discussion: Small CCTs have induced positive behavioral change and are a powerful tool for improving immunization coverage and compliance within LMICs.

Title: Clients’ perception on quality of routine immunization services for children at an urban health training centre of Delhi, India.

Background: Parental satisfaction with pediatric care is an indicator of provider quality that has been relatively unexplored in relation to childhood immunization. Studies done in various parts of the world to assess determinants and coverage of immunization have primarily focused on the deficiencies on the beneficiaries’ side; however, much less attention has been paid to factors at the health service interface in facilitating or hampering immunisation behaviour of the parents. Capturing information regarding parental satisfaction of Routine Immunization (RI) services can be used for improvement of quality which would culminate in an increase in immunization coverage.

Objectives: To assess the perceived quality, level of overall general satisfaction with RI services of clients and determine the association of factors influencing clients’ perceived quality and overall general satisfaction with RI services. 

Methods: A community based cross sectional study was conducted in an urbanized village of Delhi. A total of 279 RI visits were covered in the study, and the clients were interviewed at their residence using a pretested tool. 

Results: Dissatisfaction toward the domains of perceived quality of RI services was reported to be 3.2% for vaccine availability, 9.7% for vaccine information, 3.2% for staff behavior, 6.1% for doctor behavior, and 7.5% for infrastructure. Multivariable regression analysis indicated that distance to health facility, literacy and age of the client, doctor behavior, staff behavior, and infrastructure had an effect on overall general satisfaction of client toward RI services. 

Conclusions: The client’s perception is multidimensional; improvement in one domain is likely to strengthen the other. By understanding client’s perspective toward quality of RI service, the healthcare mangers may improve the level of overall satisfaction.

Background: Geographic clustering of high vaccine exemptions, while extensively studied and known to leave those communities vulnerable to vaccine-preventable diseases outbreaks, is also known to display great within-state heterogeneity.   The phenomena of vaccine exemption clustering has not been recently explored in Michigan. Moreover, it has not been investigated since Michigan implemented its revised nonmedical vaccine exemption policy in 2015.

Objective: This study explored vaccine exemption clustering in Michigan and examined whether nonmedical vaccine exemptions clustered by type (medical, religious, philosophical). Furthermore, the study investigated whether Michigan’s nonmedical vaccine exemption rule change had an impact on type-specific vaccine exemption clusters following its implementation.

Methods: The study used geographic information systems using the ArcGIS optimized hot spot analysis tool to visually examine vaccine exemption clustering by type in Michigan. The study analyzed school-level secondary kindergarten vaccine exemption data from individual elementary school buildings in Michigan for years spanning 2008 to 2017.

Results: Clustering of vaccine exemptions by type was present both before and after implementation of the policy with fewer statistically significant features following the policy change.  

Public Health Implications: Understanding clustering of vaccine exemptions by type can inform public health practice concerning exemption type specific vaccine interventions needed in a given area, making best use of limited resources. 

Purpose: To identify best practices for reaching vaccine-hesitant parents during face-to-face vaccine education sessions by vaccine waiver educators in Michigan.

Methods: This study utilized qualitative descriptive content analysis of semi-structured phone interviews with vaccine waiver educators from local health departments (LHDs) in Michigan. 

Results: Strategies, resources, and techniques identified by educators as beneficial included: using and providing information from a variety of sources, compiling their own educational materials, creating a positive experience, holding personalized sessions, and streamlining exemption and vaccination sessions. However, unexpected themes that emerged from the interviews indicated that vaccine waiver educators are in need of additional guidance in educating parents about vaccine ingredients, handling religious vaccine exemption requests, and assessing the role of schools. 

Discussion: Implementing successful vaccine education interventions targeting vaccine-hesitancy is crucial. Charging LHDs with overseeing vaccine education via a face-to-face discussion is a novel intervention strategy, the effective implementation of which can possibly inform vaccine education intervention nationwide and can perhaps even be translated into use in international contexts.

Introduction: Considering rapid transmission and increasing number of cases worldwide, a vaccine for coronavirus disease (COVID-19) is effective for flattening disease curve and prevention. Globally, pharmaceutical companies, university laboratories  and public health departments are working on the vaccine. Vaccination campaigns demand a percentage uptake to be effective, for COVID-19 is 55-82%. Our study suggests effective COVID-19 vaccination strategies by reviewing past successful vaccination campaigns.

Methods: This study is a review of previous successful vaccination campaigns in developing countries. We evaluate the strengths and weaknesses of each campaign to recommend the most suitable vaccination strategies for COVID-19.

Results: The goal of the COVID-19 vaccination campaign should be maximum effective coverage in the shortest timespan possible. Specific targeted mass vaccination strategies were used in response to diseases such as yellow fever epidemic, influenza outbreak and polio eradication. According to Hardt et al., targeting individuals with the highest susceptibility of acquiring risk disease or complications would be most effective. Successfully vaccinated individuals to be certified for accounting purposes. Vaccine delivery shall include school- or health center-based strategies. Infrastructure shall be developed with support from the existing systems with government ownership, supported by World Health Organization (WHO), UNICEF and Gavi. Implementation strategies involve planning, support, advocacy and communication. Voigt et al, discuss how smallpox eradication was successful with two pronged strategy of vaccination along with surveillance and containment. Polio eradication was based on four pronged strategy of routine plus extended immunization, acute flaccid paralysis surveillance and mop up vaccination rounds.

Conclusion: Mass vaccination is possible with well-coordinated area-specific approach and effective community dialogue. Risk group targeted strategy along with case surveillance in a short period of time is essential for COVID-19 disease reduction and herd protection. Local context specific communication and public-private management strategy for funding and implementation will provide a holistic picture.

Background: Influenza vaccination is recommended for all people living with HIV (PLHIV). Few data are available about coverage and modalities in this population.

• Methods:  Cross-sectional, observational and prospective study. We proposed to each HIV positive patient visiting our department of infectious diseases in Tourcoing Hospital (North, France) to fill a self-survey between the 1^st March and the 22th March 2020. 

• Results: A total of 130 patients completed the questionnaire, with 48 female (37%) and 82 male (63%) participants with a median age of 46 years (IQ 37-54). The coverage of the influenza vaccination for the previous season (2019/2020) was 67% [n =87/130 95% confidence interval (CI) 58-76%]. Older age (p=0.026), male sex (p<0.01), flu vaccine recommendation by the infectious diseases specialist (p=0.067, not statistically significant but marked trend) and generally favorable positions for vaccines (p<0.01) were associated with a positive influenza vaccination status.
The main reasons for declining influenza vaccination were fear of side effects (10/43, 23%), bad personal experience of previous influenza vaccination (10/43, 23%), and consideration as a mild illness (5/43, 12%)

• Conclusions: A correct influenza vaccination rate of 67 % was found in our HIV-positive cohort. It remains below the national recommendations. We must reinforce this practical incentive.
 Keywords: HIV, influenza vaccine, vaccine, influenza

Background: Despite the global imperative to ‘leave no child behind’, one in every 5 children remain deprived of life-saving vaccines. Low immunization coverage in part is due to the weak linkages between community and health system, inaccessibility of outsiders to closely-knit societies and low parental awareness regarding immunizations.

The Kiran Sitara (Shining Stars; KS) Adolescent Health and Leadership Program (AHLP), was implemented using adolescent girls (Kiran Sitaras-KSs) as the agents of change, who by virtue of belonging to the community were able to cause a ripple effect in boosting uptake for WHO-recommended immunizations for children under 2 years of age. Our research evaluated the feasibility of the KS community mobilization and validated KS’s ability to accurately identify under-immunized and zero-dose (never-vaccinated) children.  

Methods: AHLP was implemented in grades 8-10 of two public secondary schools in Shikarpur and Lakhi Town, Pakistan. From April-May, 2017, a door-to-door survey was conducted by KSs followed by visits of trained staff to verify the immunization statuses identified by KSs.

Results: KSs (N=45) screened 207 children and identified under-immunized and zero-dose children with an accuracy of 74% and 94% respectively. 

Discussion: Our strategy of mobilizing girls from within the communities provides a promising avenue to provide immunization services in high-risk areas that cannot be penetrated through conventional mechanisms. We are actively scaling-up our sustainable solution by implementing AHLP in 250+ schools and creating a force of 100,000+ KSs who can promote health equity today and champion the cause in their capacity as tomorrow’s mothers.

Background: COVID-19 pandemic is sweeping the world, with increasingly high morbidity and mortality rates. Vaccination is the most prominent medical option to combat the pandemic. This study aims to explore the public’s reasons to accept or reject the future COVID-19 vaccine. Methods: A web-based, cross-sectional study designed to survey Iraqi people anonymously through a self-administered questionnaire from 17- to 31 July 2020. The snowball sampling technique was recruited through social media platforms across the country. A descriptive statistic was performed using SPSS version 16. Results: A total of 406 respondents were responsible for himself, or share the responsibility, in making decisions about family members' vaccinations. More than half (221, 54.4%) were females, married (211, 52.0%), higher educated (253, 62.3%), and in the age group (18-34 years). Of the 406 respondents, 286(70.5%) interested to accept the coronavirus vaccine when it is available. Most of the respondents believed that vaccination is important to protect their families (79.3%). They considered vaccine is safe if it is tried and proven effective in other countries (88.9%). However, the vaccine should be free (76.1%). Reasons to reject vaccines were mainly related to lack of information (60.3%) about the vaccine, insufficient research has been done(81.8%), and the afraid of side effects (72.4%). Moreover, respondents think that the vaccine will be very expensive and there is no government support in Iraq (69.5%).Conclusion: Knowing the motives and barriers to accept coronavirus vaccination helps to understand the public trend when planning a local or international vaccination program to address the pandemic.

Background: Coverage with HPV vaccine is low in France, despite proven effectiveness on HPV infections and precancerous cervical lesions. WHO recommends the use of social marketing tools to mitigate vaccine hesitancy. Using a single profile discrete choice experiment (DCE), we aimed to identify optimal wording regarding characteristics of the vaccine.

Methods: Adolescents in grade 4 and 3 (aged 13-15 years) of preselected middle schools in four French regions participated in an in-class web survey in February-March 2020. In 10 hypothetical scenarios, participants decided for or against signing up for a school-based vaccination campaign against an unnamed disease. Scenarios varied by levels of four main attributes: the disease against which the vaccine protects, vaccine safety, potential for indirect protection, information on vaccine uptake among peers. Sexual transmission was included as an additional sensitive attribute.

Results: According to preliminary data, 1458 adolescents (53.5 % of girls) participated and theoretically accepted vaccination in 80.1 % of scenarios. All attributes significantly impacted theoretical vaccine acceptance. Compared to an acute febrile infectious disease, protection against cancer was motivating (odds ratio (OR) 1.29, 95%CI: 1.09-1.52), but not against genital warts (OR 0.91, 0.78-1.06). Compared to risk negation (“vaccine does not cause serious side effect”), reference to a benefit-risk balance was strongly dissuasive (OR 0.30, 0.24-0.36) and reference to ongoing international pharmacovigilance without any confirmed effect was not significantly dissuasive (OR 0.86, 0.71-1.04). The potential for indirect protection motivated acceptance, especially if high coverage could help eliminating the disease (compared to no indirect protection, OR 1.41, 1.19-1.67). Compared to the notion of insufficient coverage, reporting that >80% of youths in other countries are vaccinated motivated vaccine acceptance (OR 1.94 (1.61-2.35). Explicit mention of a sexually transmittable infection did not influence acceptance.

Conclusion: HPV vaccine communication to adolescents can be tailored to optimise the impact of promoting efforts.

Background:  

Despite sporadic skeptical denial, since its inception, vaccines have always been immensely advantageous for mankind. Even a simple thought of vaccines permeates a sense of safety against the fears and terrors of diseases, more so in case of ongoing COVID-19. Unfortunately, authenticity is shadowed by a variety of uncertified public thoughts and ignited by human propensity towards hesitancy and fears. This has been buttressed by vaccine hesitancy being listed in WHO top ten threats to global health. Besides COVID-19 has a considerable effect on vaccine compliance and coverage. Therefore, the primary aim is to attempt a comprehensive study of the impact of COVID-19 on it. 

Methodology- A literature search was performed in various databases like PubMed, Google Scholar, UpToDate, WHO and CDC websites. 

Results-According to the CDC, with the spread of COVID-19, around 114 million children from 38 countries may have missed out on the MMR vaccine excluding children who aren’t able to receive their other follow up vaccinations. About 50% of the US population weren’t given seasonal influenza vaccination in 2019. The numbers may be feared to decline further in the times of COVID-19. Contrarily,according to a survey,67% of the study population in the US agreed to take the vaccination if recommended.  

Conclusion-Influence of COVID-19 may add fuel to the fire of vaccine hesitancy due to the fear of its long-term adverse effects and unwillingness to visit hospitals. On the other hand,it may improve vaccine administration due to heightened awareness about the benefits of vaccines and the fear of pandemic. Only time will reveal the impact of COVID-19 on vaccine hesitancy, but educating people through healthcare workers, government authorities and media to increase awareness and dismissing misleading theories on vaccination will help combat this deadly virus alongside with prevention of other outbreaks.  

Vaccine hesitancy pose great challenges to the achievement of immunization targets in many countries around the world. Factors contributing to vaccine hesitancy remain largely unclear and are not well documented in many African countries. Mainly, these factors significantly affect uptake of vaccination services, and more broadly maternal, newborn and child health (MNCH) services. Parents’ risk Perceptions associated with vaccines among parents, sociocultural and economic factors” have been found to influence parents’ decisions on vaccination by many scholars predominantly from the developed countries. Therefore, there remains a paucity of information on factors contributing to vaccine hesitancy particularly, among low income mothers living in informal and rural settlements in Kenya. This study hopes to fill that gap. The value of this study is in the fact that it broadens the knowledge base on vaccine hesitancy and vaccine coverage. In order to achieve high vaccination coverage and develop strategies that will reduce vaccine hesitancy and improve vaccination uptake - factors contributing to vaccine hesitancy need to be better understood. This ongoing study seeks to establish the determinants of vaccine hesitancy, the risk perceptions associated with vaccines  and how these two variables influence decision making over vaccines, among parents living in informal settlements and rural parts of Kenya. The study employs a descriptive design exploiting a qualitative approach to study the experiences of the mothers in three counties in Kenya: Nairobi, Kiambu, and Machakos. Mothers with children under 5 years of age were purposively sampled for in-depth interviews (IDIs) and Focus group discussions (FGDs). A total of 36 IDIs (twelve IDIs per every county)  and one FGD, per every county were conducted. The findings of this study will offer insights on risk perception and determinants of vaccine hesitancy among parents from low income communities.

Vaccine hesitancy is growing.  Given that prescriber recommendation and community engagement are two effective ways of addressing this problem, training university students, especially those studying health-related subjects, to become Vaccination Champions is one way of addressing vaccine hesitancy as the Champions engage with their communities.  We therefore delivered such a course online. 

The course was advertised with the course requirements, which were to: (i) complete a pre-workshop questionnaire (which examined students’ knowledge, attitudes and beliefs about vaccination), (ii) attend the online workshop, (iii) conduct an action promoting vaccination and inform us, providing evidence, via a post-workshop questionnaire, which also solicited feedback about the workshop and re-examined the students’ knowledge, attitudes and beliefs about vaccination.  

Results:  70 students (mostly Pharmacy undergraduates and postgraduates at the UCL School of Pharmacy) completed the pre-workshop questionnaire, of whom 55 attended the live online workshop and 2 watched the workshop recording.  During the 2 hour workshop, Powerpoint slides were used to talk about vaccines and vaccination (e.g. nature, benefits, misconceptions) and vaccine hesitancy.  Breakout rooms and Slido were used to actively engage participants, e.g., to gauge their confidence about promoting vaccination and potential barriers.  Following the workshop, 53 students completed an action within 1-2 weeks, such as speaking with vaccine-hesitant family and friends, blogging and posting on social media. Analysis of the related pre- and post- workshop questionnaires (N=50) showed that gaps in the participants’ knowledge about vaccination and vaccine misconceptions were reduced post-workshop. Participants were also more likely to promote vaccination to friends and family (48 vs 38) and to strangers (41 vs 20), accept a Covid-19 vaccine (40 vs 30) and vaccinate themselves (44 vs 42) and their children (49 vs 50) following the workshop.

In future, we will hold such training regularly, tailored to different groups e.g. students studying non-science subjects.

Zika virus (ZIKV) reemergence poses a significant health threat especially due to its risks to fetal development, necessitating safe and effective vaccines that can protect pregnant women. Zika envelope domain III (ZE3) has been identified as a safe and effective vaccine candidate, however, it is poorly immunogenic. We have previously shown that plant-made recombinant immune complex (RIC) vaccines that consist of an antibody fused, via the C-terminus, to a desired antigen followed by an antibody-specific epitope tag, are a robust platform to improve the immunogenicity of weak antigens. In this study, we altered the antigen fusion site on the RIC platform to accommodate an N-terminal fusion to the IgG heavy chain (N-RIC), and thus a wider range of antigens, with a resulting 40% improvement in RIC expression over the normal C-terminal fusion (C-RIC). Both types of RICs containing ZE3 were efficiently assembled in plants and purified to >95% homogeneity with a simple one-step purification. Both ZE3 RICs strongly bound complement receptor C1q and elicited strong ZE3-specific antibody titers that correlated with ZIKV neutralization. When either N-RIC or C-RIC was co-delivered with plant-produced hepatitis B core (HBc) virus-like particles (VLP) displaying ZE3, the combination elicited 5-fold greater antibody titers (>1,000,000) and more strongly neutralized ZIKV than either RICs or VLPs alone, after only two doses without adjuvant. These findings demonstrate that antigens that require a free N-terminus for optimal antigen display can now be used with the RIC system and that plant-made RICs and VLPs are highly effective vaccines targeting ZE3. Thus, the RIC platform can be more generally applied to a wider variety of antigens.

Arboviruses have become a major public health problem in the tropical and subtropical regions of the world. The Zika virus (ZIKV) is an arboviral disease prevalent in the Americas, Africa and Asia and has increased its area of endemicity and it is considered a major public health problem in our country.  Due to the advances of the infections caused by the ZIKV it is of extreme importance the development of tools that allow the adequate combat to the ZIKV. Virus-like particles (VLPs) vaccines appear as an enormous potential for use as extremely effective antiviral vaccines, since they mimic the viral particle, inducing immune response and, as they don’t have the genetic material of the virus they won’t replicate making them safe as viral particles. In this work, we established a methodology for production and characterization of VLPs containing the structural proteins C, prM and E of ZIKV produced in insect cells using the gene expression system derived from baculovirus.  In order to obtain the recombinant baculovirus (BV-ZIKV), vectors containing the sequences of the proteins of interest were constructed. Infection assays were performed with a multiplicity of viral infection (MOI) of 2 of the inoculum of BV-ZIKV. Sf-9 cells were infected and were collected at 96h post-infection. The expression of protein E in the cells superficies could be observed by immunofluorescence and western blot assays. To concentrate and purify the VLPs from the BV-ZIKV we tested the iodixanol gradient and  we could evaluate the correct expression of E-ZIKV proteins by SDS-PAGE and western blot. The VLPs were analyzed by transmission electron microscopy and we could observe structures of 30 to 60nm in size that resembled the native ZIKV. The results of this project can generate important tools in the development of a vaccine method against the ZIKV.

Rapid and durable protection against Chikungunya is highly desired, but currently no licensed vaccine exists. An unadjuvanted Chikungunya virus-like particle (CHIKV VLP) candidate vaccine has previously demonstrated a good safety profile and robust immunogenicity in phase 1 and 2 trials in both CHIKV-naive and CHIKV-exposed adults. Here we report interim results of a phase 2 trial of alum-adjuvanted CHIKV VLP. Healthy US adults 18 to 45 years of age (n=415) were given a 1- or 2-dose series at doses of 6 to 40 mcg over a 2- or 4-week period. Serum neutralizing antibody (SNA) was assessed by a luciferase-based assay and 80% neutralization titers (NT80s) were calculated. All regimens were well-tolerated, with mostly mild or moderate injection site pain in 21% to 49% of subjects, and no vaccine-related severe (Grade 3 or higher) or serious adverse events or discontinuations. Seroconversion occurred in 74% to 98% of subjects by 7 days after 1 dose, and in all subjects by 28 days after the last dose (the primary endpoint). Peak NT80s were similar to those seen after natural CHIKV infection. The immune response was persistent, with mean NT80s of 196 to 457 through 6 months. Long-term follow-up is ongoing. These findings strongly support the potential utility and continued development of an alum-adjuvanted VLP-based vaccine for the prevention of CHIKV infection, including future studies in a wider age range.

Flaviviruses are mostly arbovirus and many of them produce human diseases of major public health importance, as Yellow Fever (YFV), Dengue (DENV) and Zika (ZIKV) viruses. Their genome encodes a single polyprotein, which is cleaved to form structural (C, prM and E) and non-structural (Ns1-Ns5) proteins. In Brazil ZIKV was introduced in 2015, causing a dengue-like epidemic that was further associated with a congenital syndrome affecting the central nervous system resulting in newborn microcephaly. Thus, the development of a protective vaccine against Zika virus infections is of high importance.  Therefore, our aim is to develop a recombinant chimeric vaccine against ZIKV. The whole genome of an engineered recombinant virus has been cloned into a pCC1 plasmid vector under T7 promoter control. The viral construct (YFV17D/ZIKV) includes the backbone of YFV strain 17D with its structural prM and E genes exchanged by the respective genes of ZIKV. The viral recovery was realized by transfecting the plasmid containing YFV17D/ZIKV with Lipofectamine into BSR-T7/5 cells, and a plasmid containing GFP under T7 promoter was used as positive control. The formation of viral RNA was evaluated in 1 and 7 d.p.t., by RT-qPCR (from viral RNA) or by qPCR (from plasmid DNA) of the supernatant and cell samples. To confirm the production of viral proteins, an immunofluorescence (IFA) of the transfected product with DAPI and YFV polyclonal antibody (in house mouse hyperimmune serum) was performed. Our results show an efficient transfection and production of viral RNA by qPCR and RT-qPCR. The viral proteins were observed by IFA. Our next steps include confirmation of the viral production after passages of the supernatant in Vero E6 and C6/36 cells in order to determine if this chimeric virus is viable.

The coronavirus disease 2019 (COVID-19) has unequivocally affected the lives of people across the planet and has imposed an unprecedented burden on our healthcare systems. With no potent regimen for treatment, there is a dire need for finding promising candidates. Receptor binding domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has proven to be a promising target owing to its role in viral invasion. Convalescent plasma therapy is widely used against COVID-19 and other respiratory diseases caused by coronaviruses. However, such treatment modes are contingent upon the availability of donors who have survived the infection. Furthermore, given the current burden on our healthcare, convalescent plasma therapy is insufficient to cover for all patients as limited antibodies can be procured from donors. Hence, promising antibodies must be designed, which can be produced on a large-scale to meet current demands. Our study aimed at generating antibody candidates from the human antibody CR3022 (derived from convalescent SARS patient) against the RBD of SARS-CoV-2 via in silico affinity maturation. We optimized the paratope of the CR3022 antibody towards the RBD of SARS-CoV-2 for better binding affinity and stability, employing molecular modeling, docking, and dynamics simulations. Out of seven antibody leads generated post in silico site-directed mutagenesis followed by preliminary screening, lead antibody named SAM3 was predicted to have the highest binding affinity towards RDB. However, molecular dynamics simulation of fifty nanoseconds set the seal on leads SAM1 and SAM2. Both demonstrated a higher binding affinity and stability compared to other counterparts and CR3022. We hypothesize that SAM1, SAM2, and SAM3 can bind to the RBD and potentially disrupt the viral invasion. Our promising study calls for further in vitro and in vivo testing of SAM1, SAM2, and SAM3 candidates for COVID-19 treatment.

Brucella is a facultative intracellular pathogen that causes a worldwide zoonotic infection known as brucellosis. Animal infections are characterised by reproductive disorders, abortions, stillbirths, infertility and milk excretion. Humans commonly acquire the infection by direct contact with infected livestock or by consumption of unpasteurised products. Brucella melitensis generally the most frequently occurring bacteria, which preferred hosts are small ruminants. Since no safe vaccines exist for humans, animal vaccination is an essential tool to control the dissemination of the pathogen. Rev1 is the only vaccine recommended for sheep and goats. However, this vaccine has several drawbacks, in particular, Rev1 infects placentas and induces abortions when administered during pregnancy. As alternative to Rev1, we have developed Rev1∆wzm and 16M∆wzm deletion mutants in the two-component ABC transport system of O-Polysaccharide (O-PS), thus, showing rough phenotype and cytoplasmic accumulation of O-PS. Both strains have exhibited excellent in vitro and in vivo vaccine properties, with clear differences between them. Due to the marked Brucella tropism for placentas, we investigated the ability of both mutants to colonize and replicate in trophoblast cells and mice placentas/foetuses, in comparison to 16M and Rev1 strains. As result, Dwzm mutants showed reduced replication in immortalised trophoblasts and mice placentas/foetuses along pregnancy. In mice, both 16M and Rev1 strains replicated actively in placentas/foetuses, inducing foetal reabsorption, stillbirth and/or new-born animals highly infected. In contrast, ∆wzm mutants were found in placentas only at a very low rate and only during the first days post-infection, clearing quickly and allowing the normal course of pregnancies and healthy uninfected placentas and foetuses; histologically, placental discs and chorionic envelopes were intact. Also, the strains tested induced different cytokine patterns that could explain the dynamic of the placental infections and clearance. These results suggest promising safety properties of Rev1∆wzm and 16M∆wzm as alternatives to Rev1.

The ongoing pandemic of infectious Coronavirus disease (COVID-19) caused by a novel SARS-CoV-2, poses a serious global health emergency. This positive-sense single-stranded RNA virus falls under the Betacoronavirus genus Coronaviridae family.  Still, now, no approved vaccine is available to combat the severity of this disease. RNA Dependent RNA Polymerase (RdRp) is an enzyme solely responsible for SARS-CoV-2 genome replication, multiplication, and has been described as a potential target for drugs. In this study, using in silico analysis we suggest candidate drugs that can specifically bind with the viral protein RdRp leading to the inhibition of genome replication. The sequence data of RdRp were retrieved from the NCBI database. Protein modeling was done with the SwissModel database. Sequences were then used to search potential drugs from Drug Bank which suggested 8 drugs. Among them, seven showed a promising binding affinity to our target protein RdRp between -8 Kcal/mole to above. Their binding efficiency was determined with Molecular Docking Analysis using Auto Dock tools-1.1.5. Among these seven drugs, Remdesivir (DB14761) which is already approved by FDA, found to have the highest binding affinity (-16 Kcal/mole) followed by 2- [(2,4-dichloro-5-methyl phenyl) Sulfonyl]-1,3-Dinitro-5-(Trifluoromethyl) Benze (DB07620) (-13.1 Kcal/mole) and Nalpha-[(benzyloxy)carbonyl]-N-[(1R)-4-hydroxy-1-methyl-2-oxobutyl]-L-phenylalaninamide (DB08732) (-13 Kcal/mole). Although, Remdesivir (DB14761) already found to have limitations in treatment against SARS-CoV-2. Our in silico analysis elucidated instead of Remdesivir, these newly identified six drugs can be used as lead components and targets for further in vitro and in vivo inhibition studies of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 pathogen, has led to a global pandemic claiming lives and posing a serious threat to public health and social cum physical interactions. The SARS-CoV-2 ORF1ab polyprotein plays crucial role in viral RNA replication. To evaluate the mutational landscape and conserved regions of this important domain, 511 complete SARS-CoV-2 ORF1ab protein sequences mined from the NCBI viral repository from affected patients across all regions were analysed. Regions of origin are as follows Asia – 175, the Americas – 146, Europe – 135, Oceania/Australia – 44, and Africa – 11. Multiple sequence alignment of the 511 SARS-CoV-2 protein sequences was performed using ClustalX version 2.1. High recurrent mutations T265I and P765S in non-structural protein 2 (nsp2), L3606F in nsp6, P4715L in RNA dependent RNA polymerase, and P5828L and Y5865C both in nsp13 with the 3CLpro region well conserved across regions were observed. Comparative analysis of the viral sequences reveals the predominance of T265I, P5828L and Y5865C mutations in the USA with low frequencies in Europe and Asia, while P4715L mutation is predominant in Africa, Europe and the USA. Mutations are central to viral transmissibility, evolution and survival, which help them to invade host immunity and develop drug resistance. It is important to understand the mutational spectra of SARS-CoV-2 genome across regions; this will help in identifying specific genomic sites as targets for drug design and vaccine development, monitoring the spread of the virus and unraveling its evolution. 

Keywords: Coronavirus, conserved regions, multiple sequence alignment, mutations, ORF1ab sequence, SARS-CoV-2

Mycobacterium bovis BCG, a live attenuated tuberculosis vaccine offers protection against disseminated TB in children. BCG exhibits heterologous protective effects against unrelated infections and reduces infant mortality due to non-mycobacterial infections. Recent reports have suggested that BCG vaccination might have protective effects against COVID-19, however it is highly unlikely that BCG vaccine in its current form can offer complete protection against SARS-CoV-2 infection due to the lack of specific immunity. Nonetheless, recombinant BCG strains expressing antigens of SARS-CoV-2 may offer protection against COVID-19 due to the activation of innate as well as specific adaptive immune response. Further proven safety records of BCG in humans, its adjuvant activity and low cost manufacturing makes it a frontrunner in the vaccine development to stop this pandemic. In this review we discuss about the heterologous effects of BCG, induction of trained immunity and its implication in development of a potential vaccine against COVID-19 pandemic. 

(https://doi.org/10.1016/j.cellimm.2020.104187)

COVID-19, a new respiratory infection outbreak has gripped many countries and continues to affect and kills millions of people worldwide. We aimed to design a potential epitope-based subunit vaccine against SARS-CoV-2 using immunoinformatics, and reverse vaccinology approaches. Potential T and B-cell epitopes were predicted from conserved region of five immunogenic proteins (nsp7-10 and orf1b) of SARS-CoV-2. Further, these epitopes were proposed for the international therapeutic peptide vaccine. The design of the multi-epitope vaccine was built using HTLs and CTLs epitopes from five different proteins along with adjuvant to enhance the immune response. These epitopes are bonded to each other by short peptide linkers. The vaccine also carries potential B-cell linear and discontinuous epitopes, and Interferon-γ inducing epitopes. The constructed multi-epitope vaccine was found to be antigenic, nonallergic, nontoxic, stable, and cover large human population worldwide. The vaccine construct was modeled, refined, and validated by various bioinformatics tools to achieve a high-quality 3D structure. The best 3D model was applied for docking studies with toll-like receptor-3 depicts the generation of an immune response. The immune-simulation study suggested a significant increase of B-cells, Th cells, Tc cells, and IgG for the vaccine. Codon adaptation and in silico expression were also performed into the pET-28a (+) plasmid vector to check biological stability. This engineered chimeric vaccine peptide could simultaneously elicit humoral and cell-mediated immune responses. We hope to confirm this new universal predicted vaccine for clinical experiments and considered for both in vitro and in vivo studies to fight against COVID-19.

Background and purpose: In 2019, the world has witnessed the emergence of a virus that causes acute respiratory distress syndrome in human, with high mortality rates (approximately 3.7%). So far, there has been no effective treatment of COVID-19. In this study, we are going to design a multi-epitope vaccine combining several T-cell and B-cell epitopes of the SARS-CoV-2.

Materials and methods: Based on immunoinformatics strategies, B-cell and T-cell epitopes were predicted by using immune Epitope Database and Analysis Resource (IEDB) server, then the appropriate predicted epitopes were joined to each other by suitable linkers, and multi-epitope vaccine constructed was suggested as a vaccine candidate against SARS-CoV-2.

Results: In this study, 28 B-cell epitopes and 33 T-cell epitopes were predicted. Then, to design the multi epitope vaccine, 5 epitopes were used from the virion surface of spike protein and one epitope was used from intravirion region of the Envelope, Membrane and Nucleocapsid proteins that later on were joined with flexible glycine linker.

Conclusion: Based on the immunoinformatics results obtained, it seems that different epitopes from SARS-CoV-2 structural proteins have a high ability to stimulate humoral and cellular immune responses, so the multi-epitope vaccine designed with these epitopes, can help to accelerate the production of effective vaccines against COVID-19.

Background: In December 2019, an outbreak of pneumonia was caused by coronavirus 2019 (COVID-19); declared by the World Health Organization (WHO) a pandemic on March 11, 2020. The live attenuated measles-mumps-rubella (MMR) vaccine is recommended for children by the WHO and Centers for Disease Control and Prevention. This study aims to assess the possibility of an available vaccine limiting viral spread.

Methods: A systematic review of MMR vaccination and trained innate immunity in reducing the incidence and severity of COVID-19 was performed. The primary search was conducted with Medline, Pubmed, Embase, Scopus, Google Scholar, Web of Science, and CINAHL, using keywords: trained immunity, and COVID-19, MMR, and COVID-19, SARS-Cov-2 and, MMR vaccine, Pediatric vaccination. English, published, and unpublished literature was included.

Results: Based on homology sequencing, Sidiq et al. note 30 amino acid (AA) residues similar between the Spike (S) glycoprotein of COVID-19 with the fusion glycoprotein of measles, and the envelope glycoprotein of rubella. Anbarasu et al. stated that COVID-19 S glycoprotein is around 32% similar to measles and mumps. Rubella on the other hand shares 29% (Young et. al) or 33% (Anbarasu et al.) AA sequence identity with COVID-19 (See Table 1.) 

Figure 1. Percent similarities between protein components of Covid-19 and Measles, Mumps, Rubella

Young et al. demonstrates this with elevated levels of rubella IgG in COVID-19 patients. Sanjay and Sarath mentioned a milder version of COVID-19 in early childhood due to MMR vaccination. Moreover, Sidiq et al. noted South Korean, Italian, and Chinese children were less affected by the disease. According to Fidel et al., MMR vaccination in immunocompetent and non-pregnant adults can prevent deadly consequences. Deshpande and Balaji warn that well-designed controlled studies are needed before acknowledging MMR vaccination to prevent COVID-19. Randomized controlled trials are ongoing.

Conclusion: MMR may not prevent COVID-19 infection but it could potentially reduce poor outcomes especially in at-risk age groups.

The Ebola virus (EV) belongs to the Filoviridae family; the Zaire Ebola virus is primarily responsible for the Ebola virus disease. EV is a non-segmented negative RNA strand. The vaccine candidates in current progress are Vectored Vaccines based on Vesicular Stomatitis Virus (VSV-EBOV), human (Ad5, and Ad26) Chimpanzees (ChAd3) Adenoviruses, and DNA based vaccines. In a randomized clinical trial (NCT02376426), two heterologous vaccination, Ad26ZEBOV, and MVA-BN-FILO, were administrated to 60 out of 72 volunteers. The vaccines act by binding and neutralizing the Glycoprotein of EV. This trial established that these two vaccines are safe, well-tolerated, and possess immunogenic action towards the disease. 

The Nipah Virus is a potential zoonotic pathogen belonging to the Paramyxoviridae family. The fruit bats  (Pteropus spp.)  act as a natural host reservoir of the NiV. This viral disease leads to chronic encephalitis and respiratory illnesses both in humans and animals. At present biologic HeV-sG-V (NCT04199169) is in phase 1 clinical trial for testing the safety and immunogenicity of the vaccine.

The focus is mainly on novel Coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 ( SARS-CoV-2) belonging to the Betacoronavirus gene of the Coronaviridae family. This virus is an enveloped single-stranded RNS virus consisting of Spike (S), Membrane (M), Envelope (E), and Nucleocapsid (N) proteins. The virus acts via Angiotensin-Converting Enzyme 2 (ACE 2) on the host cells leading to fatal respiratory illness. A total of 211 vaccine candidates are currently in the pipeline for CoVID-19 in the form of Inactivated vaccines, Subunit vaccine, Nucleic acid-based on DNA, or mRNA vaccines, of which leading ones are the ChAdOx1 nCov-19 vaccine (NCT04324606), mRNA-1273 vaccine (NCT04283461), BNT162 vaccine (NCT04380701).

In this poster, various vaccines in the pipeline for the aforementioned viral diseases will be discussed, which possibly prevent and control epidemic/pandemic like this in the future.

Despite the availability of effective and safe vaccines for the protection of humans and animals, rabies remains a widespread disease worldwide, being transmitted by domestic and wild animals. Rabies is still responsible for the deaths of approximately 66,000 people a year worldwide and most deaths occur in countries that are still unable to produce modern vaccines or use less effective. Rabies virus encodes 5 proteins, the most important research interests have been rabies virus glycoprotein (RVGP) and matrix protein (RVM). RVGP is the major antigen capable of conferring neutralizing immune response against a rabies virus infection. Over the past three decades, virus-like particles have evolved to become a widely accepted technology, especially in the field of vaccinology. Among the numerous methods for producing VLPs, we highlight the use of the recombinant baculovirus system. Baculoviruses have been widely used in this area because they are easy to handle, safe and inexpensive. The overall objective of this project is to produce virus-like particles (VLPs) and to evaluate the expression of recombinant rabies Lyssavirus glycoprotein (RVGP) by Sf9 baculovirus system on 100 to 2000mL scales. We determine the ideal rates of cell growth and death. We also quantified limiting substrate consumption and toxic metabolite production. We determined the best physiological and biokinetic conditions of cell culture and viral infection. We established the best multiplicity of infection (MOI) and the best collection times. We expressed rabies virus VLPs and were able to quantify and qualify them by Western, Dot, ELISA and electron microscopy. Thus, this work has allowed to show the efficiency in expressing the RVM and RVGP proteins from the infection of recombinant BVs in Sf-9 cells. Our next objective is to scale production in bench bioreactor batch system.

Leptospirosis, caused by pathogenic and intermediate species of the genus Leptospira, is a disease with a high impact in the livestock industry, where it generates considerable economic losses and public health challenges due to zoonosis. As many as 15 different pathogenic or intermediate Leptospira species are known to cause the disease, however, their characterization and correct classification is essential for understanding the epidemiological role of the disease. Classification and phylogenetic analysis of a region of the rrs and rpoB genes from 55 isolates collected from across Colombia from different biological origins (humans, bovine, pigs, canines, rodents and environmental waters) was carried out. An approximation to serovar was performed by Southern blot ribotyping complemented by Multiple-Locus Variable-number tandem repeat Analysis (MLVA), using two VNTR schemes. Results were further complemented by whole genome sequencing of a representative from each of the major clades. Phylogenetic analysis of the isolates revealed three major clades: Pathogenic (29 isolates) showing 87–91% similarity to known species such as L. interrogans, L. santarosai and L. kirschneri. The second clade grouped 24 isolates of intermediate pathogenicity and the third group included only two saprophytic isolates. Genetic patterns similar to the reference serovars L. biflexa serovar Patoc, L. interrogans serovars Pomona, Canicola, copenhageni and Grippotyphosa, and a characteristic pattern of intermediate species, was found with the Southern blot ribotyping and MLVA techniques. Genome analysis further confirmed clade classification and highlighted the presence of intermediate species as causative agents of disease in the county. This is the first report suggesting high prevalence of intermediate species involved in pathology in Colombia. 

Leptospira is the causative agent of leptospirosis, the most common zoonotic disease worldwide, with a high incidence in public health and domestic animal production systems. The diagnosis of leptospirosis is complicated due to the diversity of the genus, classified into 65 species and more than 300 pathogenic, intermediate or saprophyte serovars. In host-leptospiral interaction studies, proteins – especially Outer Membrane Proteins (OMPs) that mediate the entry, spread and evasion of the immune system, have become good candidates for vaccines and diagnostic tools. However, bacterial cell growth and further purification of OMPs for characterization can be challenging. In the present study, we compared protocols for cell fractionation, using the nonionic detergents Triton X-100 and X-114 for Colombian Leptospira isolates previously classified as pathogenic and intermediate. Furthermore, solubilization buffers Urea-NaH₂PO₄-Tris and Tris-SDS, were analyzed in the aqueous and detergent phases of the Triton X-114 protocol. Resulting protein profiles were quantified by NanoDrop and visualized by SDS-PAGE 15%. A standardized protocol was obtained for the extraction of OMPs in Leptospira spp. using 2% Triton X114-10mM CaCl₂ plus Tris-SDS buffer, an essential step in proteomic studies aiming to develop vaccine immunogens or diagnostic kits that allow improving disease prevention and control measures.

Zoonoses include infectious diseases, usually transmitted between domestic or wild animals and humans. Rabies is a fatal neurological zoonotic disease caused by Lyssavirus, a bullet shaped virus affecting the central nervous system (CNS). About 55,000-60,000 individuals die of rabies annually around the world. Rabies is certainly the infection with a 100% case-fatality rate that can transfer by the bite of rabid animals to humans and animals. Domestic and wild animals such as cats, bats, foxes, monkeys and raccoons can spread this virus to humans but dogs are the leading source of rabies in human population. Pakistan is also in the list of the countries where Rabies is endemic and both livestock and human population have a great risk of this fatal disease. Generally, it creates disturbing circumstances in the nation and requires rapid intention to control the rabies and dog bite cases. Immunization is the best control strategy to fight against rabies. Huge number of individuals are experiencing rabies after being bitten by dogs in the rustic territories. Moreover, a decent number (71%) of unfortunate victims went for otherworldly treatment or neighborhood medication men. There are several constraints in rabies vaccinology in rural areas of Pakistan, as many peoples do not believe efficacy of rabies vaccine owing to improper cold chain storage system mainly due to unavailability and energy crises in these rural areas. Therefore, mindfulness programs for the locals ought to be begun at the earliest opportunity.

Brucella melitensis is the global pathogenic species of Brucella that constitutes a serious hazard to public health. Control and eradication programs have been implemented in countries where brucellosis exists. Within the strategy of control and prevention of this brucella, a new way approach, by vaccinating Iranian sheep with Rev.1 ocular vaccination is suggested. The purpose of this study was to evaluate subcutaneous and ocular route of vaccination with Rev.1 strain against B. melitensis infection in Iranian sheep.

Thirty-six brucella-free sheep obtained from Kordan research center aged 4-10 months old randomly divided to six identical groups from A to F. Efficacy of Rev.1 vaccine was investigated by two methods including; subcutaneous and ocular inoculation also two control groups were used. Groups of A and B were used for normal subcutaneous injected with different doses of Rev.1 vaccine. Groups D and E were used for ocular inoculation. Group C and F were used as control. Only group D had a booster dose six month after the first injection. The half of all groups were challenged 12 months later. The other halves of entire groups were challenged 17 month later. Serological responses were evaluated using modified RB, SA and 2MEtests.

The groups which were injected via ocular, did not show any considerable sera titre after two months, however they showed a good protection after the challenge. The group which received subcutaneous injection showed titre after 4 months. Two of the animals belong to this group even showed titre in over 6 months period. Comparison between the groups with and without booster dose showed no differences in term of protection; however, the group with no booster dose seemed to be more practical and economical for immunization. Hence it is recommended that more investigation is needed with more animal   population to come to any definite conclusion.

Efficacy of a new live Salmonella Typhimurium vaccine candidate attenuated by radiation mutation technology was evaluated in this study. Live form of the vaccine and formalin-inactivated form were orally administered. 15 piglets were used and divided into 3 groups, A to C, containing 5 piglets per group. Serum IgG titers and levels of serum IFN-γ, IL-4 of the immunized groups, especially group B significantly increased. Furthermore, no clinical signs were observed in group B piglets during the entire experimental period after the challenge, while diarrhea was observed in many of the piglets in groups A and C. No Salmonella was isolated from fecal samples of the group B piglets on day 7 after challenge, whereas the challenge strain was isolated from all and some piglets in groups A and C. These results indicate that oral vaccination of the piglets with the vaccine induced effective protection against Salmonella infections in the growing piglets.

The present study investigates the enhancement of immunogenicity and protection efficacy of Salmonella Typhimurium ghosts surface-displayed with FliC against chicken salmonellosis. The membrane-anchored FliC is a potential TLR-5 agonist, delivers an essential adjuvant effect for the ghost vaccine candidate. The present ghost plasmid pJHL184 construct carries a convergent dual promoter system that has the temperature-dependent induction of the phage lysis gene E and the target antigen FliC at the same time. Under permissible conditions of temperatures, less than 30 ^oC at the presence of 20 mM L-arabinose effectively suppresses expression of the lysis gene. Once the temperature is up-lifted to 42 ^oC without arabinose, cause the generation of ST ghosts expelling the cytoplasmic content. The addition of FliC adjuvant significantly enhanced the IgY response, cell-mediated immune responses, regulatory cytokine induction and subsequently enhanced protection against Salmonella challenge. Further, intramuscular immunization with ST ghosts displaying FliC induced particularly high CD8+ response demarcating its proficiency to elicit Type I immune responses. Further, ST ghosts displaying FliC caused an increase in both CD4+ and CD8+ response compared to the PBS control suggesting its capability to engage both cell-mediated and humoral immune responses essential for the elimination of Salmonella. Upon the virulent challenge, we could observe a significant reduction in challenged bacterial load on spleen and liver tissues in the ST ghosts surface-displaying FliC adjuvant. Our study suggests the biological incorporation of FliC on ST ghosts enhances vaccine immunogenic potency and acts as a safe and effective prevention strategy against chicken salmonellosis.

In the present study, we developed an O-antigen-deficient, live, attenuated Salmonella Typhimurium (ST) strain (JOL2377) and assessed its safety, macrophage toxicity, invasion into lymphoid tissues, immunogenicity, and protection against Salmonella infection in chickens. The JOL2377 induced significantly lower cytotoxicity and higher inflammatory cytokines IL-2, IL-10, IL-4, and IFN- γ responses than the WT strain upon macrophage uptake. It did not persist in macrophages or in chicken organs and rapidly cleared without systemic infection. None of the chicken were found to secrete Salmonella in feces into the environment exacerbating its attenuation. Interestingly JOL2377 did not deprive for arrival in immunological hot-spots such as spleen, liver and bursa of Fabricius for an efficient antigen presentation and immune stimulation. Mucosal and parenteral immunization with JOL2377 significantly elicit antigen-specific humoral (IgY) and cell mediated responses marked by peripheral blood mononuclear cell proliferation, cytokine induction, increase in T-cell responses than non-immunized control. JOL2377 did not generate significant levels of LPS specific antibodies as compared to the WT strain due to the lack of immunogenic O-antigen component from its LPS structure. Upon virulent challenge, route dependent efficacy differences were leaving the intramuscular route is superior to the oral route on reducing splenic and liver colonization of the challenge ST. The least cytotoxicity, virulence, and superior immunogenicity of JL2377 that effectively engage both humoral and IFN- γ mediated CMI responses present an ideal scenario in host immune modulation to fight against intracellular pathogen Salmonella.

The development of a safe vaccine against Salmonella would be important for both farm animals and humans with regard to disease containment. The SE ghosts carrying FliC were genetically constructed using a special ghost plasmid pJHL184 that co-expresses FliC and the phage lysis gene E. These SE ghosts were characterized by ghost generation efficacy by increasing the culture temperature to 42^oC in the absence of L-arabinose. This temperature change led to an efficient ghost generation with almost complete lysis of the SE host strain. The expression of FliC was confirmed by Western blot analysis. In addition, direct ELISA was used to prove FliC specific antibody generation in immunized mice. The parenteral adjuvant effect of the FliC antigen was demonstrated by immunizing mice with pJHL184::flC, pJHL184 alone or PBS alone. The mice were intramuscularly immunized at 6 weeks of age (n=8) and boosted after 3 weeks of primary inoculation. A total of 32 mice were equally divided into four groups. Each group was treated with pJHL-ghosts alone, ghost surface displaying FliC adjuvant, and compared to the PBS and naïve control groups. The immunized mice demonstrated greater IgG and IgA antibody responses than did the PBS control group. Furthermore, the addition of the ghosts to the FliC led to a significant increase in both the humoral and cell mediated immune responses compared to those in the ghost alone group. This result revealed the adjuvant efficacy. In addition, the in vitro antigen uptake and presentation studies revealed very efficient antigen presentation on the mouse macrophage cell surfaces. This finding further corroborated the potential efficacy of immune stimulation. After the virulent challenge, we observed a significant reduction in the bacterial load in the spleen and liver tissues in SE ghosts surface displaying FliC adjuvant. 

Despite extensive vaccination, enzootic outbreaks of H9N2 avian influenza have often occurred and resulted in great economic losses in the poultry industries. The aim of this study was to carry out genetic and antigenic analyses of Iranian H9N2s during 2010-2018 and compare them with the current vaccine seed strain. From 2010 to 2018, samples were collected from broilers suffering from severe respiratory signs and high mortality. In the current study, 51 broiler farms out of 233 were positive for H9N2 viruses. Of 51 viruses, 12 (2017-2018) and three (2010-2013) H9N2s were selected for final genetic and antigenic characterization. The HA and NA genes of 15 H9N2s and a common vaccine strain were completely sequenced and compared with other representative Iranian viruses. The recent isolates shared low sequence identity in both HA (89%) and NA (90%) genes with the vaccine strain. Phylogenetic analysis revealed that all Iranian viruses were grouped into G1 sub-lineage with different clusters in which recent isolates (2014-2017) formed a distinct cluster compared to the vaccine group (1998-2004). Four independent antigenic groups which had low antigenic reactivity with current vaccine strain were distinguished using web-based AntigenMap analysis software. Recent isolated H9N2 viruses formed a novel antigenic group and displayed more than 3-fold change in the HI assay (P<0.001). In the immune protection evaluation, the titers of H9N2 viruses in the tracheal and cloacal swabs of candidate vaccine strain were significantly lower than the current vaccine and the control groups (P<0.05) on day 5 and 7 post infection (PI). At 9 days PI, no virus shedding was observed in the tracheal and cloacal samples in either recommended or current vaccine groups. The findings of this study highlight that currently used vaccine strain requires updating to the most appropriate virus in order to achieve optimal protection against H9N2 viruses.

The Sars-Cov-2 that caused the Covid-19 pandemic has caused great concern among health and population authorities around the world. The virus recognizes human cells through the spike protein, specifically from the receptor-binding domain (RBD), making these regions important alternatives for vaccine development. In this work, it was expressed, and it was determined the immunogenicity of RBD protein from Sars-Cov-2 in the Baculovirus expression system.

Cultures of 7 liters of Sf-9 cells were infected at a density of 3x10⁶ cells/mL at an infection multiplicity (MOI) of 3. After 48 hours of infection, the culture medium was centrifuged, the supernatant collected and concentrated through tangential filtration using a 10 KDa MWCO membrane. Afterward, there were carried out two purification steps by affinity chromatography and molecular exclusion, the purity obtained was proved by SDS-PAGE. ELISA affinity assay was performed to test the integrity of the purified protein. To test the immunogenicity of the protein, ELISA assay was performed using sera of convalescent patients.

RBD productivity levels of up to 0.6 mg/liter at >95% purity were achieved and the binding EC50 to the human ACE-2 receptor was 46.8 ng/mL. The final purified RBD was recognized by a panel of five convalescent sera. This antigen could be a candidate for use in future animal trials to test its immunogenicity, as well as its toxicity and observe for adverse reactions in Phase I and II clinical trials.

Brucellosis is a worldwide bacterial zoonosis that implies enormous economic losses, since livestock is the main reservoir and the disease is clinically characterized by at term abortion outbreaks, perinatal mortality and sterility in animals. Human disease mostly occurs through direct contact with infectious material and consumption of raw milk and derivatives. Currently, there are no safe vaccines for humans and treatments are tedious and quite often ineffective, leading to recurrences and permanent sequelae. The preferable strategy to prevent human brucellosis is to control the infection in animals, particularly Brucella melitensis in sheep and goats. Rev1 is the only recommended vaccine against the virulent infection in small ruminants. However, Rev1 owns some drawbacks, i.e. pathogenicity in pregnant ewes, infective for human beings, resistance to streptomycin (treatment of choice for human brucellosis) and interference in serological diagnosis, recommending the development of a safer vaccine. We have developed the Rev1Δwzm vaccine candidate through a targeted and irreversible deletion of wzm gene, involved in the two-component ABC transport system responsible for the lipopolysaccharide O-chain export to the periplasmic space. As expected, Rev1Δwzm showed rough phenotype and cytoplasmic accumulation of O-polysaccharide. Also, the vaccine candidate was susceptible to streptomycin, cationic antimicrobial peptides and serum complement from the innate immune system. Besides, it was sensitive to several environmental factors, such as desiccation, oxidative stress, acidic pH, temperature, nutrient deprivation and surfactant compounds. On the other hand, in mice, Rev1Δwzm was highly attenuated, did not infect placentas and induced transient splenomegaly, associated with the triggering of an effective adaptive response. In fact, Rev1Δwzm have proved as effective as Rev1 against B. melitensis virulent infection in mice. Overall, Rev1Δwzm is a promising vaccine candidate as alternative to Rev1 to be studied in the natural host.

Rabies is a zoonotic disease responsible for more than 60.000 human deaths annually. The rabies affects different species of mammals. The virus encodes 5 proteins, for this work we used matrix protein (RVM) and glycoprotein (RVGP). The RVGP is the only protein exposed on the virus surface and it is able to induce neutralizing antibodies that make it interesting to be expressed in many systems. It is assumed that RVM constitutes major structural components of the rabies virus, and it has an important role in virus assembly and budding. The focus of this paper is to produce recombinant baculovirus bearing RVM and RVGP proteins and to produce VLPs that are able to inducing immune response. We constructed 2 recombinant baculovirus, one containing the matrix protein gene (BVM) and the second containing the rabies glycoprotein gene (BVG). We coinfected Sf9 cells using MOI 0.1 for each of them. We collected the cells and supernatant 48 h and 96 h post coinfection. The supernatant was clarified and concentrated by sucrose gradient. The samples were analyzed by: ELISA, negative stain and electronic microscopic. The sucrose gradient allowed us to separate the VLPs from the BVs. We can detect the VLPs in ≈ 40% of sucrose gradient and the BVs detect in the pellet. ELISA method allowed us to dose the RVGP protein at different stages of purification of rabies VLPs. The cells were visualized by transmission electron microscopy, we can visualize BVs structure into cell nucleus, and also budding from cell membrane. We also observed, on the external side from the cell, structures to resemble to the rabies VLPs. These assays were efficient for production and purification of VLPs. In our studies, we were able to separate VLPs from recombinant BVs and we can quantify the total RVGP in the supernatant.