Virus Challenge Research Articles

Antiviral effect of Favipiravir against Chandipura virus in vitro and in vivo.

Chandipura virus (CHPV) is an emerging encephalitic virus with outbreak potential in a pediatric population. It causes acute encephalitis, with clinical symptoms leading to death within 48-72 h and an alarmingly high case fatality rate up to 55%-78%. Despite the high mortality rate in children, no vaccines or antivirals are currently available; thus, repurposing licensed drugs seems to be one of the attractive therapeutic approaches. Among the various options available, Favipiravir emerged as a promising candidate, and its unique characteristics and clinical efficacy have garnered significant attention and demonstrated considerable potential in the fight against viral diseases. In the current study, we have evaluated the antiviral effect of Favipiravir against CHPV by Plaque reduction assay and viral growth kinetics assay in Vero cells and in vivo effect of drug treatment against lethal viral challenge was analysed in 10-day-old CD1 mice. A dose-dependent reduction in CHPV plaque size and number was observed in Vero cells treated with Favipiravir, with an EC50 of 92.26 μM. Complete inhibition of CHPV replication was observed at 320 μM drug concentration and 50% cytotoxicity (CC50 ) at 4774 μM, indicating a high selectivity index 51.24. In vivo, studies in mice showed 100% survival with 300 mg/kg/day of Favipiravir given orally till seventh-day postinfection. The study provides evidence of the antiviral activity of Favipiravir against CHPV infection, and further clinical evaluation may alleviate the associated mortality.

Highly lethal genotype I and II recombinant African swine fever viruses detected in pigs

African swine fever virus (ASFV) poses a great threat to the global pig industry and food security. Currently, 24 ASFV genotypes have been reported but it is unclear whether recombination of different genotype viruses occurs in nature. In this study, we detect three recombinants of genotype I and II ASFVs in pigs in China. These recombinants are genetically similar and classified as genotype I according to their B646L gene, yet 10 discrete fragments accounting for over 56% of their genomes are derived from genotype II virus. Animal studies with one of the recombinant viruses indicate high lethality and transmissibility in pigs, and deletion of the virulence-related genes MGF_505/360 and EP402R derived from virulent genotype II virus highly attenuates its virulence. The live attenuated vaccine derived from genotype II ASFV is not protective against challenge of the recombinant virus. These naturally occurring recombinants of genotype I and II ASFVs have the potential to pose a challenge to the global pig industry.

Beta-containing bivalent SARS-CoV-2 protein vaccine elicits durable broad neutralization in macaques and protection in hamsters

BackgroundSince the beginning of the COVID-19 pandemic, several variants of concern (VOC) have emerged for which there is evidence of an increase in transmissibility, more severe disease, and/or reduced vaccine effectiveness. Effective COVID-19 vaccine strategies are required to achieve broad protective immunity against current and future VOC.MethodsWe conducted immunogenicity and challenge studies in macaques and hamsters using a bivalent recombinant vaccine formulation containing the SARS-CoV-2 prefusion-stabilized Spike trimers of the ancestral D614 and the variant Beta strains with AS03 adjuvant (CoV2 preS dTM-AS03) in a primary immunization setting.ResultsWe show that a primary immunization with the bivalent CoV2 preS dTM-AS03 elicits broader and durable (1 year) neutralizing antibody responses against VOC including Omicron BA.1 and BA.4/5, and SARS-CoV-1 as compared to the ancestral D614 or Beta variant monovalent vaccines in naïve non-human primates. In addition, the bivalent formulation confers protection against viral challenge with SARS-CoV-2 prototype D614G strain as well as Alpha and Beta variant strains in hamsters.ConclusionsOur findings demonstrate the potential of a Beta-containing bivalent CoV2 preS dTM-AS03 formulation to provide broad and durable immunogenicity, as well as protection against VOC in naïve populations.

A Booster with a Genotype-Matched Inactivated Newcastle Disease Virus (NDV) Vaccine Candidate Provides Better Protection against a Virulent Genotype XIII.2 Virus.

Newcastle disease (ND) is endemic in Bangladesh. Locally produced or imported live Newcastle disease virus (NDV) vaccines based on lentogenic virus strains, locally produced live vaccines of the mesogenic Mukteswar strain, as well as imported inactivated vaccines of lentogenic strains, are being used in Bangladesh under different vaccination regimens. Despite these vaccinations, frequent outbreaks of ND are being reported in Bangladesh. Here we compared the efficacy of booster immunization with three different vaccines in chickens that had been primed with two doses of live LaSota vaccine. A total of 30 birds (Group A) were primed with two doses of live LaSota virus (genotype II) vaccine at days 7 and 28, while 20 birds (Group B) remained unvaccinated. At day 60, birds of Group A were divided into three sub-groups, which received booster immunizations with three different vaccines; A1: live LaSota vaccine, A2: inactivated LaSota vaccine, and A3: inactivated genotype XIII.2 vaccine (BD-C161/2010 strain from Bangladesh). Two weeks after booster vaccination (at day 74), all vaccinated birds (A1-A3) and half of the unvaccinated birds (B1) were challenged with a genotype XIII.2 virulent NDV (BD-C161/2010). A moderate antibody response was observed after the primary vaccination, which substantially increased after the booster vaccination in all groups. The mean HI titers induced by the inactivated LaSota vaccine (8.0 log2/5.0 log2 with LaSota/BD-C161/2010 HI antigen) and the inactivated BD-C161/2010 vaccine (6.7 log2/6.2 log2 with LaSota/BD-C161/2010 HI antigen) were significantly higher than those induced by the LaSota live booster vaccine (3.6 log2/2.6 log2 with LaSota/BD-C161/2010 HI antigen). Despite the differences in the antibody titers, all chickens (A1-A3) survived the virulent NDV challenge, while all the unvaccinated challenged birds died. Among the vaccinated groups, however, 50% of the chickens in Group A1 (live LaSota booster immunization) shed virus at 5- and 7-days post challenge (dpc), while 20% and 10% of the chickens in Group A2 (inactivated LaSota booster immunization) shed virus at 3 and 5 dpc, respectively, and only one chicken (10%) in Group A3 shed virus at 5 dpc. In conclusion, the genotype-matched inactivated NDV booster vaccine offers complete clinical protection and a significant reduction in virus shedding.

Hemagglutinin virus-like particles incorporated with membrane-bound cytokine adjuvants provide protection against homologous and heterologous influenza virus challenge in aged mice

BackgroundCurrent influenza vaccines deliver satisfactory results in young people but are less effective in the elderly. Development of vaccines for an ever-increasing aging population has been an arduous challenge due to immunosenescence that impairs the immune response in the aged, both quantitatively and qualitatively.ResultsTo potentially enhance vaccine efficacy in the elderly, we investigated the immunogenicity and cross-protection of influenza hemagglutinin virus-like particles (HA-VLP) incorporated with glycosylphosphatidylinositol (GPI)-anchored cytokine-adjuvants (GPI-GM-CSF and GPI-IL-12) via protein transfer in aged mice. Lung viral replication against homologous and heterologous influenza viruses was significantly reduced in aged mice after vaccination with cytokine incorporated VLPs (HA-VLP-Cyt) in comparison to HA-VLP alone. Enhanced IFN-γ+CD4+ and IFN-γ+CD8+ T cell responses were also observed in aged mice immunized with HA-VLP-Cyt when compared to HA-VLP alone.ConclusionsCytokine-adjuvanted influenza HA-VLP vaccine induced enhanced protective response against homologous influenza A virus infection in aged mice. Influenza HA-VLP vaccine with GPI-cytokines also induced enhanced T cell responses correlating with better protection against heterologous infection in the absence of neutralizing antibodies. The results suggest that a vaccination strategy using cytokine-adjuvanted influenza HA-VLPs could be used to enhance protection against influenza A virus in the elderly.

ISCOMs/MPLA-Adjuvanted SDAD Protein Nanoparticles Induce Improved Mucosal Immune Responses and Cross-Protection in Mice.

The epidemics caused by the influenza virus are a serious threat to public health and the economy. Adding appropriate adjuvants to improve immunogenicity and finding effective mucosal vaccines to combat respiratory infection at the portal of virus entry are important strategies to boost protection. In this study, a novel type of core/shell protein nanoparticle consisting of influenza nucleoprotein (NP) as the core and NA1-M2e or NA2-M2e fusion proteins as the coating antigens by SDAD hetero-bifunctional crosslinking is exploited. Immune-stimulating complexes (ISCOMs)/monophosphoryl lipid A (MPLA) adjuvants further boost the NP/NA-M2e SDAD protein nanoparticle-induced immune responses when administered intramuscularly. The ISCOMs/MPLA-adjuvanted protein nanoparticles are delivered through the intranasal route to validate the application as mucosal vaccines. ISCOMs/MPLA-adjuvanted nanoparticles induce significantly strengthened antigen-specific antibody responses, cytokine-secreting splenocytes in the systemic compartment, and higher levels of antigen-specific IgA and IgG in the local mucosa. Meanwhile, significantly expanded lung resident memory (RM) T and B cells (TRM /BRM ) and alveolar macrophages population are observed in ISCOMs/MPLA-adjuvanted nanoparticle-immunized mice with a 100% survival rate after homogeneous and heterogeneous H3N2 viral challenges. Taken together, ISCOMs/MPLA-adjuvanted protein nanoparticles could improve strong systemic and mucosal immune responses conferring protection in different immunization routes.

Homologous Sequential Immunization Using Salmonella Oral Administration Followed by an Intranasal Boost with Ferritin-Based Nanoparticles Enhanced the Humoral Immune Response against H1N1 Influenza Virus.

The influenza virus continues to pose a great threat to public health due to the frequent variations in RNA viruses. Vaccines targeting conserved epitopes, such as the extracellular domain of the transmembrane protein M2 (M2e), a nucleoprotein, and the stem region of hemagglutinin proteins, have been developed, but more efficient strategies, such as nanoparticle-based vaccines, are still urgently needed. However, the labor-intensive in vitro purification of nanoparticles is still necessary, which could hinder the application of nanoparticles in the veterinary field in the future. To overcome this limitation, we used regulated lysis Salmonella as an oral vector with which to deliver three copies of M2e (3M2e-H1N1)-ferritin nanoparticles in situ and evaluated the immune response. Then, sequential immunization using Salmonella-delivered nanoparticles followed by an intranasal boost with purified nanoparticles was performed to further improve the efficiency. Compared with 3M2e monomer administration, Salmonella-delivered in situ nanoparticles significantly increased the cellular immune response. Additionally, the results of sequential immunization showed that the intranasal boost with purified nanoparticles dramatically stimulated the activation of lung CD11b dendritic cells (DCs) and elevated the levels of effector memory T (TEM) cells in both spleen and lung tissues as well as those of CD4 and CD8 tissue-resident memory T (TRM) cells in the lungs. The increased production of mucosal IgG and IgA antibody titers was also observed, resulting in further improvements to protection against a virus challenge, compared with the pure oral immunization group. Salmonella-delivered in situ nanoparticles efficiently increased the cellular immune response, compared with the monomer, and sequential immunization further improved the systemic immune response, as shown by the activation of DCs, the production of TEM cells and TRM cells, and the mucosal immune response, thereby providing us with a novel strategy by which to apply nanoparticle-based vaccines in the future. IMPORTANCE Salmonella-delivered in situ nanoparticle platforms may provide novel nanoparticle vaccines for oral administration, which would be beneficial for veterinary applications. The combination of administering Salmonella-vectored, self-assembled nanoparticles and an intranasal boost with purified nanoparticles significantly increased the production of effector memory T cells and lung resident memory T cells, thereby providing partial protection against an influenza virus challenge. This novel strategy could open a novel avenue for the application of nanoparticle vaccines for veterinary purposes.

Self-injury and viral challenges among adolescents

The use of the internet to search for information or share images about self-harm is an emerging risk among young people. Additionally, viral challenges involving engaging in physical self-harm and posting it on the internet are becoming increasingly prevalent. This conference analyzed the prevalence of online self-harm and viral challenges, as well as differences by gender and age. The relationship between online self-harm and intrapersonal factors (depression and anxiety) and interpersonal factors (family cohesion, social resources) were also examined. A sample of 1,877 adolescents (946 females) between the ages of 12 and 17 (mean age = 13.41, SD = 1.255) who completed self-report measures were evaluated. According to the results obtained, it was presented that 11% of adolescents had engaged in some form of online self-harm. Prevalence was significantly higher among females and adolescents over the age of is Depression and anxiety increased the risk of online self-harm, while family cohesion was a protective factor against it. In summary, the presentation emphasized that the use of the internet to share or seek information about self-harm is a problem that involves a significant proportion of adolescents. Online risk prevention programs should include emotional regulation, coping, and resilience skills to reduce the potential negative consequences of these behaviors.

Whole‐genome sequencing identifies interferon-induced protein IFI6/IFI27-like as a strong candidate gene for VNN resistance in European sea bass

BackgroundViral nervous necrosis (VNN) is a major disease that affects European sea bass, and understanding the biological mechanisms that underlie VNN resistance is important for the welfare of farmed fish and sustainability of production systems. The aim of this study was to identify genomic regions and genes that are associated with VNN resistance in sea bass.ResultsWe generated a dataset of 838,451 single nucleotide polymorphisms (SNPs) identified from whole-genome sequencing (WGS) in the parental generation of two commercial populations (A: 2371 individuals and B: 3428 individuals) of European sea bass with phenotypic records for binary survival in a VNN challenge. For each population, three cohorts were submitted to a red-spotted grouper nervous necrosis virus (RGNNV) challenge by immersion and genotyped on a 57K SNP chip. After imputation of WGS SNPs from their parents, quantitative trait loci (QTL) were mapped using a Bayesian sparse linear mixed model (BSLMM). We found several QTL regions that were specific to one of the populations on different linkage groups (LG), and one 127-kb QTL region on LG12 that was shared by both populations and included the genes ZDHHC14, which encodes a palmitoyltransferase, and IFI6/IFI27-like, which encodes an interferon-alpha induced protein. The most significant SNP in this QTL region was only 1.9 kb downstream of the coding sequence of the IFI6/IFI27-like gene. An unrelated population of four large families was used to validate the effect of the QTL. Survival rates of susceptible genotypes were 40.6% and 45.4% in populations A and B, respectively, while that of the resistant genotype was 66.2% in population B and 78% in population A.ConclusionsWe have identified a genomic region that carries a major QTL for resistance to VNN and includes the ZDHHC14 and IFI6/IFI27-like genes. The potential involvement of the interferon pathway, a well-known anti-viral defense mechanism in several organisms (chicken, human, or fish), in survival to VNN infection is of particular interest. Our results can lead to major improvements for sea bass breeding programs through marker-assisted genomic selection to obtain more resistant fish.

Efficient ex vivo expansion of conserved element vaccine-specific CD8+ T-cells from SHIV-infected, ART-suppressed nonhuman primates.

HIV-specific T cells are necessary for control of HIV-1 replication but are largely insufficient for viral clearance. This is due in part to these cells' recognition of immunodominant but variable regions of the virus, which facilitates viral escape via mutations that do not incur viral fitness costs. HIV-specific T cells targeting conserved viral elements are associated with viral control but are relatively infrequent in people living with HIV (PLWH). The goal of this study was to increase the number of these cells via an ex vivo cell manufacturing approach derived from our clinically-validated HIV-specific expanded T-cell (HXTC) process. Using a nonhuman primate (NHP) model of HIV infection, we sought to determine i) the feasibility of manufacturing ex vivo-expanded virus-specific T cells targeting viral conserved elements (CE, CE-XTCs), ii) the in vivo safety of these products, and iii) the impact of simian/human immunodeficiency virus (SHIV) challenge on their expansion, activity, and function. NHP CE-XTCs expanded up to 10-fold following co-culture with the combination of primary dendritic cells (DCs), PHA blasts pulsed with CE peptides, irradiated GM-K562 feeder cells, and autologous T cells from CE-vaccinated NHP. The resulting CE-XTC products contained high frequencies of CE-specific, polyfunctional T cells. However, consistent with prior studies with human HXTC and these cells' predominant CD8+ effector phenotype, we did not observe significant differences in CE-XTC persistence or SHIV acquisition in two CE-XTC-infused NHP compared to two control NHP. These data support the safety and feasibility of our approach and underscore the need for continued development of CE-XTC and similar cell-based strategies to redirect and increase the potency of cellular virus-specific adaptive immune responses.

Resilience of swine nasal microbiota to influenza A virus challenge in a longitudinal study

Influenza A virus (IAV) is an important contributing pathogen of porcine respiratory disease complex (PRDC) infections. Evidence in humans has shown that IAV can disturb the nasal microbiota and increase host susceptibility to bacterial secondary infections. Few, small-scale studies have examined the impact of IAV infection on the swine nasal microbiota. To better understand the effects of IAV infection on the nasal microbiota and its potential indirect impacts on the respiratory health of the host, a larger, longitudinal study was undertaken to characterize the diversity and community composition of the nasal microbiota of pigs challenged with an H3N2 IAV. The microbiome of challenged pigs was compared with non-challenged animals over a 6-week period using 16S rRNA gene sequencing and analysis workflows to characterize the microbiota. Minimal changes to microbial diversity and community structure were seen between the IAV infected and control animals the first 10 days post-IAV infection. However, on days 14 and 21, the microbial populations were significantly different between the two groups. Compared to the control, there were several genera showing significant increases in abundance in the IAV group during acute infection, such as Actinobacillus and Streptococcus. The results here highlight areas for future investigation, including the implications of these changes post-infection on host susceptibility to secondary bacterial respiratory infections.

Intermediate levels of preexisting protective antibody allow priming of protective T cell immunity against Influenza

Abstract Overcoming interfering impacts of preexisting immunity to generate universally protective influenza A virus (IAV)-specific T cell immunity through vaccination is a high priority. Here, we passively transfer varied amounts of H1N1-IAV-specific immune serum prior to H1N1-IAV infection to determine how different levels of preexisting Ab influence the generation and protective potential of heterosubtypic T cell responses in a murine model. Surprisingly, IAV nucleoprotein (NP)-specific CD4 and CD8 T cell responses are readily detected in infected recipients of IAV-specific immune serum regardless of the amount transferred. When compared to responses in control groups and recipients of low and intermediate levels of convalescent serum, NP-specific T cell responses in recipients of high levels of IAV-specific serum, which prevent overt weight loss and reduce peak viral titers in the lungs, are, however, markedly reduced. While detectable at priming, this response recalls poorly and is unable to mediate protection against a lethal heterotypic (H3N2) virus challenge at later memory timepoints. A similar failure to generate protective heterosubtypic T cell immunity during IAV priming is seen in offspring of IAV-primed mothers that naturally receive high titers of IAV-specific Ab through maternal transfer. Our findings support that priming of protective heterosubtypic T cell responses can occur in the presence of intermediate levels of preexisting Ab. These results have high relevance to vaccine approaches aiming to incorporate and evaluate cellular and humoral immunity against IAV and other viral pathogens against which T cells can protect against variants escaping Ab-mediated protection. Supported grants from NIH (RO1 AI165406) Startup funds from the University of Central Florida foundation

SARS-CoV-2 Ancestral and Variant of Concern Breakthrough infections boost both cross-reactive cellular and humoral immune responses in mRNA-vaccinated Hamsters.

Abstract When mRNA vaccines were rolled out under EUA, it was observed that a single vaccination, already protected vaccinees from severe disease, even before neutralizing antibodies appearded. To better understand the underlying mechanism of protection, we vaccinated hamsters once with a suboptimal dose of Pfizer/BioNTech COVID-19 mRNA vaccine (5mg/animal). Four weeks post vaccination, animals were challenged with different variants of SARS-CoV-2. Vaccination resulted in antibody ELISA titers against SARS-CoV-2 spike protein, but antibody titers were too low for efficient neutralization of antigenically matching USA-WA1/2020 (WA1) or drifted variants of concern (Alpha, Beta, Delta and Mu). Nevertheless, suboptimal vaccination resulted in reduced morbidity for WA1 and Alpha-challenged animals and complete control of lung virus titers for WA1, Alpha and Delta but with breakthrough infection for the antigenically more distant Beta and Mu. Moreover, T cell responses were higher in vaccinated animals compared to unvaccinated ones at 5 days post infection, suggesting a recall of vaccine-primed T cells. Infection with different SARS-CoV-2 variants also back-boosted neutralizing antibody responses against the challenge virus and in the case of the variants of concern, against the antigenically distant but vaccine-matched WA1. Transcriptomic analysis of host immune responses to infection reflects both vaccination status and disease course, is further compared with lung pathology and suggests a role for interstitial lung macrophages in vaccine-mediated protection. In summary, protection after suboptimal vaccination against SARS-CoV-2 challenge in hamsters correlates with recall of both vaccine-induced B- and T-cell responses. This study was also partly funded by CRIPT (Center for Research on Influenza Pathogenesis and Transmission), a NIH NIAID funded Center of Excellence for Influenza Research and Response (CEIRR, contract number 75N93021C00014) to A.G.S and M.S. and by R01 AI160706 to M.S.

Pandemic preparedness: Addition of TLR agonists CpG+MPLA to emulsified H5 confers protection against H5N1 challenge after a single dose.

Abstract H5N1 vaccines adjuvanted in the oil-in-water emulsion MF59 are maintained in the Strategic National Stockpile for pre-pandemic preparedness. The US population are currently naïve to H5 and a prime/boost regimen will likely be needed to engender a protective response. Ideally, a rapid response to pandemic influenza would benefit from being able to induce protection from a single dose of vaccine. Here, we have used a systems immunology approach in mice to examine the effect of formulating recombinant H5 in AddaVax alone or AddaVax with CpG+MPLA. AddaVax emulsion alone does not stimulate the innate immune system, although the oil droplets do act as a nanocarrier for antigen and enhance B cell activation, presumably by BCR cross-linking. In vitro, germinal center reactions show inclusion of CpG+MPLA to the emulsion (a combination adjuvant we call IVAX-1) synergize to enhance B cell maturation and class-switching. Similarly, in vivo, the combination of H5/IVAX-1 enhances both H5-specific B and T cell responses compared to individual components. Single cell mRNA sequencing of spleen cells after a boost indicate responses to H5/CpG+MPLA and H5/IVAX-1 are more similar to each other but distinct from H5/AddaVax or H5/PBS. Unexpectedly, H5/AddaVax, H5/CpG+MPLA and H5/IVAX-1 all engendered 100% efficacy against lethal intranasal challenge of H5N1 virus, although only H5/IVAX-1 engendered 100% protection after a single dose. Our data indicate the potential for certain combination adjuvants to enhance the immunogenicity of influenza vaccines and achieve efficacy from single dose vaccination. Supported by AI160397. NIAID Grant AI160397

Single-cell atlas of the liver myeloid compartment with cure of chronic viral hepatitis

Abstract Background/Aims: Chronic viral infections present serious public health challenges. Direct-acting antivirals (DAAs) are now able to cure nearly all patients infected with the hepatitis C virus (HCV), representing the only cure of a human chronic viral infection to date. DAAs provide a valuable opportunity to study immune pathways in the reversal of chronic immune failures in humans. Methods: To leverage this opportunity, we used plate-based single-cell RNA-seq (scRNA-seq) to deeply profile myeloid cells from liver fine needle aspirates in HCV patients before and after DAA treatment. We characterized liver neutrophils, eosinophils, mast cells, conventional dendritic cells (cDCs), plasmacytoid DCs, classical/non-classical monocytes, and macrophages, and defined fine-grained subpopulations of several cell types. Results: We discovered cell-type-specific changes post-cure, including an increase in MCM7+STMN1+proliferating CD1C+ cDCs, which may support restoration from chronic exhaustion. We observed an unexpected inverse relationship between pre-treatment viral load and post-cure ISG expression in each cell type, revealing a link between viral loads and sustained modifications of the host’s immune system. We found an upregulation of PD-L1 expression in ISG-high neutrophils, pinpointing cell subpopulations crucial for immune regulation. We identified three recurring gene programs shared by multiple cell types, distilling core functions of the myeloid compartment. Conclusion: This comprehensive scRNA-seq atlas of human liver myeloid cells in response to a cure of chronic viral infections reveals principles of liver immunity and provides immunotherapeutic insights. The single-cell sorting was done at the MGH Flow and Mass Cytometry Research Core, which was supported by NIH Shared Instrumentation program grants 1S10OD012027-01A1, 1S10OD016372-01, 1S10RR020936-01, and 1S10RR023440-01A1. Additional sorting was done at the Immunology Core Facility of the Ragon Institute, which is part of the Harvard University Center for AIDS Research (CFAR), an NIH funded program (P30 AI060354).

SARS-CoV-2 RBD-specific atypical memory B cells exhibit effector memory-like features in multi-dose mRNA vaccination

Abstract Memory B cell is a heterogenous population and such heterogeneity is a focus of active investigation. How distinct subsets of memory B cells respond to repeated novel antigen challenges such as SARS-CoV-2 in human remains a subject of vigorous research. We employed fluorochrome-conjugated streptavidin tetramers bound with RBD of wild-type and omicron variant (B.1.1.529), respectively, to sort RBD-specific B cells and performed CITE-seq to investigate detailed dynamics of transcriptomes, surface phenotyes and clonality in healthy mRNA vaccination cohort. PBMC samples of 8 BNT162b2 vaccine recipients from 7 consecutive timepoints, before and after each dose of 3-times vaccination, were analyzed. We found pulsating, yet with increasing trend, frequency of atypical memory B cells among tetramer-binding B cells after each dose of vaccine. SCENIC analysis of tetramer-binding memory B cells to find subset-specific regulatory gene networks revealed atypical memory B cells have distinct regulons of genes related to activation such as receptor clustering, signaling and calcium-dependent activation compared with classical memory B cells and found only one shared clone between atypical and classical tetramer-binding memory B cells, suggesting infrequent interconversion between the two subsets. Furthermore, in-vitro polyclonal stimulation of atypical memory B cells showed higher frequency of antibody-secreting cells (ASC) and pre-plasmablasts in flow cytometry and wild-type RBD-specific ASCs in IgG ELISPOT assay, compared with classical memory B cells. These findings highlight effector memory-like features of atypical memory B cells in viral antigen challenge and a potential target of future vaccine design.

Iterative hypothesis testing in HIV vaccine research: moving towards success.

Iterative hypothesis testing in HIV vaccine research: moving towards success.

ISCOMs/MPLA adjuvanted SDAD protein nanoparticles induce improved immune responses and cross-protection in mice

Abstract The development of a universal influenza vaccine to protect against influenza variants will be the priority goal in combating influenza virus infection. In this study, we exploited a novel type of protein nanoparticles consisting of influenza NP as the core and NA1-M2e or NA2-M2e fusion proteins as the coating antigens by using an SDAD heterobifunctional crosslinker. Intramuscular immunization of the NP/NA-M2e (core/shell) nanoparticles induced increased humoral and cellular immune responses. ISCOMs/MPLA as adjuvants further improved the nanoparticle immunogenicity and elicited stronger immune reactions conferring protection against different influenza viral challenges. The nanoparticles were delivered through intranasal route to determine the application as a mucosal vaccine. While the nanoparticles alone barely induced immune response after intranasal immunization, ISCOMs/MPLA adjuvanted nanoparticles induced strengthened antigens-specific antibodies responses, cellular responses and higher levels of mucosal IgA. Meanwhile, increased lung Trm, Brm , and alveolar macrophages, were observed from ISCOMs/MPLA adjuvanted nanoparticle group. Only the mice intranasally vaccinated with ISCOMs/MPLA adjuvanted nanoparticles fully survived during the infections, and better protection efficacy was observed when compared with other adjuvants. Our results emphasized the importance of supplementing of appropriate adjuvants to improve the immunogenicity and mucosal immune responses of vaccines in mucosal immunization. In conclusion, the ISCOMs/MPLA adjuvant combination could significantly improve the immune responses and protective efficiency of protein nanoparticles in different immunization routes.

Identification of novel genes that regulate B-1 cells

Abstract IgM is the first antibody produced in ontogeny and in response to an initial antigen exposure. It plays an important role in effective immune protection, and is required for maximal antigen-specific IgG responses. In mice, most circulating serum IgM is generated by fetal-derived B-1 cells in spleen and bone marrow. How B-1 cells preferentially develop into natural IgM secreting cells is incompletely understood. To identify candidate genes critical for B-1 cells development and/or maintenance we generated parts of a larger immunophenotyping database Knock Out Mouse Project (KOMP) providing results of the flow cytometric evaluation of spleen cells from 641 mouse strains, each containing a single-gene deletion as well as 200 individual congenic control mice, identifying 58 distinct leukocyte subsets for each spleen. We generated and curated a complete list of each gene found to be associated with B-1 cell changes. Seven genes were identified that significantly reduced frequencies of CD5+ B-1 cells in males and/or females, of which three were uniquely altering the B-1 cell subset. None of these genes have been associated with B-1 cell regulation previously. Using qRT-PCR we found that 4 showed B-1 cell intrinsic gene expression. Ongoing work is to determine the impact of each gene on serum antibody levels and immune protection to influenza virus challenge. This work was supported by NIH UM1 OD023221 (KCKL), R0AI148652 (NB), and the Graduate Student Support Program UC Davis (SR).

Impact of prior flavivirus vaccination on immunogenicity and efficacy of an inactivated Zika vaccine in Indian rhesus macaques.

Flaviviruses are antigenically related. We evaluated the immunogenicity and efficacy of Takeda's purified inactivated Zika vaccine (PIZV) candidate in macaques previously vaccinated with several commercially available heterologous flavivirus vaccines. Heterologous flavivirus vaccination did not elicit Zika virus (ZIKV) neutralizing antibodies and did not impact neutralizing antibody titers after one dose of PIZV. After a second PIZV dose previous vaccination with flavivirus vaccines had variable impact on ZIKV neutralizing antibody titers. However, all macaques were protected against viremia after Zika virus challenge 8-12months post-PIZV vaccination. Therefore, vaccine-induced immunity against heterologous flavivirus vaccines does not impact PIZV efficacy in macaques.