Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2 heterodimer as a vaccine candidate.

SignificanceHepatitis C virus chronically infects approximately 1% of the world's population, making an effective vaccine for hepatitis C virus a major unmet public health need. The membrane-associated E1E2 envelope glycoprotein has been used in clinical studies as a vaccine candidate. However, limited neutralization breadth and difficulty in producing large amounts of homogeneous membrane-associated E1E2 have hampered efforts to develop an E1E2-based vaccine. Our previous work described the design and biochemical validation of a native-like soluble secreted form of E1E2 (sE1E2). Here, we describe the immunogenic characterization of the sE1E2 complex. sE1E2 elicited broadly neutralizing antibodies in immunized mice, with increased neutralization breadth relative to the membrane-associated E1E2, thereby validating this platform as a promising model system for vaccine development.

Ultrapotent neutralizing antibodies against SARS-CoV-2 with a high degree of mutation resistance.

Many SARS-CoV-2 neutralizing antibodies (nAbs) lose potency against variants of concern. In this study, we developed 2 strategies to produce mutation-resistant antibodies. First, a yeast library expressing mutant receptor binding domains (RBDs) of the spike protein was utilized to screen for potent nAbs that are least susceptible to viral escape. Among the candidate antibodies, P5-22 displayed ultrahigh potency for virus neutralization as well as an outstanding mutation resistance profile. Additionally, P14-44 and P15-16 were recognized as mutation-resistant antibodies with broad betacoronavirus neutralization properties. P15-16 has only 1 binding hotspot, which is K378 in the RBD of SARS-CoV-2. The crystal structure of the P5-22, P14-44, and RBD ternary complex clarified the unique mechanisms that underlie the excellent mutation resistance profiles of these antibodies. Secondly, polymeric IgG enhanced antibody avidity by eliminating P5-22's only hotspot, residue F486 in the RBD, thereby potently blocking cell entry by mutant viruses. Structural and functional analyses of antibodies screened using both potency assays and the yeast RBD library revealed rare, ultrapotent, mutation-resistant nAbs against SARS-CoV-2.

Matrix M Adjuvanted H5N1 Vaccine Elicits Broadly Neutralizing Antibodies and Neuraminidase Inhibiting Antibodies in Humans That Correlate With In Vivo Protection.

The highly pathogenic avian influenza H5N1 viruses constantly evolve and give rise to novel variants that have caused widespread zoonotic outbreaks and sporadic human infections. Therefore, vaccines capable of eliciting broadly protective antibody responses are desired and under development. We here investigated the magnitude, kinetics and protective efficacy of the multi-faceted humoral immunity induced by vaccination in healthy adult volunteers with a Matrix M adjuvanted virosomal H5N1 vaccine. Vaccinees were given escalating doses of adjuvanted vaccine (1.5µg, 7.5µg, or 30µg), or a non-adjuvanted vaccine (30µg). An evaluation of sera from vaccinees against pseudotyped viruses covering all (sub)clades isolated from human H5N1 infections demonstrated that the adjuvanted vaccines (7.5µg and 30µg) could elicit rapid and robust increases of broadly cross-neutralizing antibodies against all clades. In addition, the adjuvanted vaccines also induced multifaceted antibody responses including hemagglutinin stalk domain specific, neuraminidase inhibiting, and antibody-dependent cellular cytotoxicity inducing antibodies. The lower adjuvanted dose (1.5µg) showed delayed kinetics, whilst the non-adjuvanted vaccine induced overall lower levels of antibody responses. Importantly, we demonstrate that human sera post vaccination with the adjuvanted (30µg) vaccine provided full protection against a lethal homologous virus challenge in mice. Of note, when combining our data from mice and humans we identified the neutralizing and neuraminidase inhibiting antibody titers as correlates of in vivo protection.

Anti-Drug Antibodies in Pigtailed Macaques Receiving HIV Broadly Neutralising Antibody PGT121.

Broadly neutralising antibodies (bNAbs) may play an important role in future strategies for HIV control. The development of anti-drug antibody (ADA) responses can reduce the efficacy of passively transferred bNAbs but the impact of ADA is imperfectly understood. We previously showed that therapeutic administration of the anti-HIV bNAb PGT121 (either WT or LALA version) controlled viraemia in pigtailed macaques with ongoing SHIV infection. We now report on 23 macaques that had multiple treatments with PGT121. We found that an increasing number of intravenous doses of PGT121 or human IgG1 isotype control antibodies (2-4 doses) results in anti-PGT121 ADA induction and low plasma concentrations of PGT121. ADA was associated with poor or absent suppression of SHIV viremia. Notably, ADA within macaque plasma recognised another human bNAb 10E8 but did not bind to the variable domains of PGT121, suggesting that ADA were primarily directed against the constant regions of the human antibodies. These findings have implications for the development of preclinical studies examining multiple infusions of human bNAbs.

Cross-Neutralization of Emerging SARS-CoV-2 Variants of Concern by Antibodies Targeting Distinct Epitopes on Spike.

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have arisen that exhibit increased viral transmissibility and partial evasion of immunity induced by natural infection and vaccination. To address the specific antibody targets that were affected by recent viral variants, we generated 43 monoclonal antibodies (mAbs) from 10 convalescent donors that bound three distinct domains of the SARS-CoV-2 spike. Viral variants harboring mutations at K417, E484, and N501 could escape most of the highly potent antibodies against the receptor binding domain (RBD). Despite this, we identified 12 neutralizing mAbs against three distinct regions of the spike protein that neutralize SARS-CoV-2 and variants of concern (VOCs), including B.1.1.7 (alpha), P.1 (gamma), and B.1.617.2 (delta). Notably, antibodies targeting distinct epitopes could neutralize discrete variants, suggesting that different variants may have evolved to disrupt the binding of particular neutralizing antibody classes. These results underscore that humans exposed to the first pandemic wave of prototype SARS-CoV-2 possess neutralizing antibodies against current variants and that it is critical to induce antibodies targeting multiple distinct epitopes of the spike that can neutralize emerging variants of concern. IMPORTANCE We describe the binding and neutralization properties of a new set of human monoclonal antibodies derived from memory B cells of 10 coronavirus disease 2019 (COVID-19) convalescent donors in the first pandemic wave of prototype SARS-CoV-2. There were 12 antibodies targeting distinct epitopes on spike, including two sites on the RBD and one on the N-terminal domain (NTD), that displayed cross-neutralization of VOCs, for which distinct antibody targets could neutralize discrete variants. This work underlines that natural infection by SARS-CoV-2 induces effective cross-neutralization against only some VOCs and supports the need for COVID-19 vaccination for robust induction of neutralizing antibodies targeting multiple epitopes of the spike protein to combat the current SARS-CoV-2 VOCs and any others that might emerge in the future.

Implications of a highly divergent dengue virus strain for cross-neutralization, protection, and vaccine immunity.

Although divergent dengue viruses (DENVs) have been isolated in insects, nonhuman primates, and humans, their relationships to the four canonical serotypes (DENV 1-4) are poorly understood. One virus isolated from a dengue patient, DKE-121, falls between genotype and serotype levels of sequence divergence to DENV-4. To examine its antigenic relationship to DENV-4, we assessed serum neutralizing and protective activity. Whereas DENV-4-immune mouse sera neutralize DKE-121 infection, DKE-121-immune sera inhibit DENV-4 less efficiently. Passive transfer of DENV-4 or DKE-121-immune sera protects mice against homologous, but not heterologous, DENV-4 or DKE-121 challenge. Antigenic cartography suggests that DENV-4 and DKE-121 are related but antigenically distinct. However, DENV-4 vaccination confers protection against DKE-121 in nonhuman primates, and serum from humans immunized with a tetravalent vaccine neutralize DENV-4 and DKE-121 infection equivalently. As divergent DENV strains, such as DKE-121, may meet criteria for serotype distinction, monitoring their capacity to impact dengue disease and vaccine efficacy appears warranted.

Broadly-Neutralizing Antibodies Against Emerging SARS-CoV-2 Variants.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have become a major concern in the containment of current pandemic. The variants, including B.1.1.7 (Alpha), B.1.351 (Beta), P1 (Gamma) and B.1.617.2 (Delta) have shown reduced sensitivity to monoclonal antibodies, plasma and/or sera obtained from convalescent patients and vaccinated individuals. Development of potent therapeutic monoclonal antibodies (mAbs) with broad neutralizing breadth have become a priority for alleviating the devastating effects of this pandemic. Here, we review some of the most promising broadly neutralizing antibodies obtained from plasma of patients that recovered from early variants of SARS-CoV-2 that may be effective against emerging new variants of the virus. This review summarizes several mAbs, that have been discovered to cross-neutralize across Sarbecoviruses and SARS-CoV-2 escape mutants. Understanding the characteristics that confer this broad and cross-neutralization functions of these mAbs would inform on the development of therapeutic antibodies and guide the discovery of second-generation vaccines.

Validation of a Triplex Pharmacokinetic Assay for Simultaneous Quantitation of HIV-1 Broadly Neutralizing Antibodies PGT121, PGDM1400, and VRC07-523-LS.

The outcome of the recent Antibody Mediated Prevention (AMP) trials that tested infusion of the broadly neutralizing antibody (bnAb) VRC01 provides proof of concept for blocking infection from sensitive HIV-1 strains. These results also open up the possibility that triple combinations of bnAbs such as PGT121, PGDM1400, as well as long-lasting LS variants such as VRC07-523 LS, have immunoprophylactic potential. PGT121 and PGDM1400 target the HIV-1 V3 and V2 glycan regions of the gp120 envelope protein, respectively, while VRC07-523LS targets the HIV-1 CD4 binding site. These bnAbs demonstrate neutralization potency and complementary breadth of HIV-1 strain coverage. An important clinical trial outcome is the accurate measurement of in vivo concentrations of passively infused bnAbs to determine effective doses for therapy and/or prevention. Standardization and validation of this testing method is a key element for clinical studies as is the ability to simultaneously detect multiple bnAbs in a specific manner. Here we report the development of a sensitive, specific, accurate, and precise multiplexed microsphere-based assay that simultaneously quantifies the respective physiological concentrations of passively infused bnAbs in human serum to ultimately define the threshold needed for protection from HIV-1 infection.

Implications of Dengue Virus Maturation on Vaccine Induced Humoral Immunity in Mice.

The use of dengue virus (DENV) vaccines has been hindered by the complexities of antibody dependent enhancement (ADE). Current late-stage vaccine candidates utilize attenuated and chimeric DENVs that produce particles of varying maturities. Antibodies that are elicited by preferentially exposed epitopes on immature virions have been linked to increased ADE. We aimed to further understand the humoral immunity promoted by DENV particles of varying maturities in an AG129 mouse model using a chimeric insect specific vaccine candidate, bDENV-2. We immunized mice with mature, partially mature, and immature bDENV-2 and found that immunization with partially mature bDENV-2 produced more robust and cross-neutralizing immune responses than immunization with immature or mature bDENV-2. Upon challenge with mouse adapted DENV-2 (D220), we observed 80% protection for mature bDENV-2 vaccinated mice and 100% for immature and partially mature vaccinated mice, suggesting that protection to homotypic challenge is not dependent on maturation. Finally, we found reduced in vitro ADE at subneutralising serum concentrations for mice immunized with mature bDENV-2. These results suggest that both immature and mature DENV particles play a role in homotypic protection; however, the increased risk of in vitro ADE from immature particles indicates potential safety benefits from mature DENV-based vaccines.

Site-Specific Glycan-Masking/Unmasking Hemagglutinin Antigen Design to Elicit Broadly Neutralizing and Stem-Binding Antibodies Against Highly Pathogenic Avian Influenza H5N1 Virus Infections.

The highly pathogenic avian influenza (HPAI) H5N1 viruses with the capability of transmission from birds to humans have a serious impact on public health. To date, HPAI H5N1 viruses have evolved into ten antigenically distinct clades that could cause a mismatch of vaccine strains and reduce vaccine efficacy. In this study, the glycan masking and unmasking strategies on hemagglutinin antigen were used for designing two antigens: H5-dm/st2 and H5-tm/st2, and investigated for their elicited immunity using two-dose recombinant H5 (rH5) immunization and a first-dose adenovirus vector prime, followed by a second-dose rH5 protein booster immunization. The H5-dm/st2 antigen was found to elicit broadly neutralizing antibodies against different H5N1 clade/subclade viruses, as well as more stem-binding antibodies to inhibit HA-facilitated membrane fusion activity. Mice immunized with the H5-dm/st2 antigen had a higher survival rate when challenged with homologous and heterologous clades of H5N1 viruses. Mutant influenza virus replaced with the H5-dm/st2 gene generated by reverse genetics (RG) technology amplified well in MDCK cells and embryonated chicken eggs. Again, the inactivated H5N1-dm/st2 RG virus elicited more potent cross-clade neutralizing and anti-fusion antibodies in sera. Therefore, the H5N1-dm/st2 RG virus with the site-specific glycan-masking on the globular head and the glycan-unmasking on the stem region of H5 antigen can be used for further development of cross-protective H5N1 vaccines.

Coronavirus Pseudotypes for All Circulating Human Coronaviruses for Quantification of Cross-Neutralizing Antibody Responses.

The novel coronavirus SARS-CoV-2 is the seventh identified human coronavirus. Understanding the extent of pre-existing immunity induced by seropositivity to endemic seasonal coronaviruses and the impact of cross-reactivity on COVID-19 disease progression remains a key research question in immunity to SARS-CoV-2 and the immunopathology of COVID-2019 disease. This paper describes a panel of lentiviral pseudotypes bearing the spike (S) proteins for each of the seven human coronaviruses (HCoVs), generated under similar conditions optimized for high titre production allowing a high-throughput investigation of antibody neutralization breadth. Optimal production conditions and most readily available permissive target cell lines were determined for spike-mediated entry by each HCoV pseudotype: SARS-CoV-1, SARS-CoV-2 and HCoV-NL63 best transduced HEK293T/17 cells transfected with ACE2 and TMPRSS2, HCoV-229E and MERS-CoV preferentially entered HUH7 cells, and CHO cells were most permissive for the seasonal betacoronavirus HCoV-HKU1. Entry of ACE2 using pseudotypes was enhanced by ACE2 and TMPRSS2 expression in target cells, whilst TMPRSS2 transfection rendered HEK293T/17 cells permissive for HCoV-HKU1 and HCoV-OC43 entry. Additionally, pseudotype viruses were produced bearing additional coronavirus surface proteins, including the SARS-CoV-2 Envelope (E) and Membrane (M) proteins and HCoV-OC43/HCoV-HKU1 Haemagglutinin-Esterase (HE) proteins. This panel of lentiviral pseudotypes provides a safe, rapidly quantifiable and high-throughput tool for serological comparison of pan-coronavirus neutralizing responses; this can be used to elucidate antibody dynamics against individual coronaviruses and the effects of antibody cross-reactivity on clinical outcome following natural infection or vaccination.

The Ability of Porcine Reproductive and Respiratory Syndrome Virus Isolates to Induce Broadly Reactive Neutralizing Antibodies Correlates With In Vivo Protection.

Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most relevant diseases of swine. The condition is caused by PRRS virus (PRRSV), an extremely variable virus of the Arteriviridae family. Its heterogeneity can be responsible, at least partially, of the poor cross-protection observed between PRRSV isolates. Neutralizing antibodies (NAs), known to play a role in protection, usually poorly recognize heterologous PRRSV isolates, indicating that most NAs are strain-specific. However, some pigs develop broadly reactive NAs able to recognize a wide range of heterologous isolates. The aim of this study was to determine whether PRRSV isolates that induce broadly reactive NAs as determined in vitro are able to confer a better protection in vivo. For this purpose two in vivo experiments were performed. Initially, 40 pigs were immunized with a PRRSV-1 isolate known to induce broadly reactive NAs and 24 additional pigs were used as controls. On day 70 after immunization, the pigs were divided into eight groups composed by five immunized and three control pigs and exposed to one of the eight different heterologous PRRSV isolates used for the challenge. In the second experiment, the same experimental design was followed but the pigs were immunized with a PRRSV-1 isolate, which is known to generate mostly strain-specific NAs. Virological parameters, specifically viremia and the presence of challenge virus in tonsils, were used to determine protection. In the first experiment, sterilizing immunity was obtained in three groups, prevention of viremia was observed in two additional groups, although the challenge virus was detected occasionally in the tonsils of immunized pigs, and partial protection, understood as a reduction in the frequency of viremia compared with controls, was recorded in the remaining three groups. On the contrary, only partial protection was observed in all groups in the second experiment. The results obtained in this study confirm that PRRSV-1 isolates differ in their ability to induce cross-reactive NAs and, although other components of the immune response might have contributed to protection, pigs with cross-reactive NAs at the time of challenge exhibited better protection, indicating that broadly reactive NAs might play a role in protection against heterologous reinfections.

Pan-Sarbecovirus Neutralizing Antibodies in BNT162b2-Immunized SARS-CoV-1 Survivors.

Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern pose a challenge to the effectiveness of current vaccines. A vaccine that could prevent infection caused by known and future variants of concern as well as infection with pre-emergent sarbecoviruses (i.e., those with potential to cause disease in humans in the future) would be ideal. Here we provide data showing that potent cross-clade pan-sarbecovirus neutralizing antibodies are induced in survivors of severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) infection who have been immunized with the BNT162b2 messenger RNA (mRNA) vaccine. The antibodies are high-level and broad-spectrum, capable of neutralizing not only known variants of concern but also sarbecoviruses that have been identified in bats and pangolins and that have the potential to cause human infection. These findings show the feasibility of a pan-sarbecovirus vaccine strategy. (Funded by the Singapore National Research Foundation and National Medical Research Council.).

Influenza virus infection expands the breadth of antibody responses through IL-4 signalling in B cells.

Influenza viruses are a major public health problem. Vaccines are the best available countermeasure to induce effective immunity against infection with seasonal influenza viruses; however, the breadth of antibody responses in infection versus vaccination is quite different. Here, we show that nasal infection controls two sequential processes to induce neutralizing IgG antibodies recognizing the hemagglutinin (HA) of heterotypic strains. The first is viral replication in the lung, which facilitates exposure of shared epitopes that are otherwise hidden from the immune system. The second process is the germinal center (GC) response, in particular, IL-4 derived from follicular helper T cells has an essential role in the expansion of rare GC-B cells recognizing the shared epitopes. Therefore, the combination of exposure of the shared epitopes and efficient proliferation of GC-B cells is critical for generating broadly-protective antibodies. These observations provide insight into mechanisms promoting broad protection from virus infection.

COVID-19 mRNA vaccine induced antibody responses against three SARS-CoV-2 variants.

As SARS-CoV-2 has been circulating for over a year, dozens of vaccine candidates are under development or in clinical use. The BNT162b2 mRNA COVID-19 vaccine induces spike protein-specific neutralizing antibodies associated with protective immunity. The emergence of the B.1.1.7 and B.1.351 variants has raised concerns of reduced vaccine efficacy and increased re-infection rates. Here we show, that after the second dose, the sera of BNT162b2-vaccinated health care workers (n = 180) effectively neutralize the SARS-CoV-2 variant with the D614G substitution and the B.1.1.7 variant, whereas the neutralization of the B.1.351 variant is five-fold reduced. Despite the reduction, 92% of the seronegative vaccinees have a neutralization titre of >20 for the B.1.351 variant indicating some protection. The vaccinees' neutralization titres exceeded those of recovered non-hospitalized COVID-19 patients. Our work provides evidence that the second dose of the BNT162b2 vaccine induces cross-neutralization of at least some of the circulating SARS-CoV-2 variants.

Prior aerosol infection with lineage A SARS-CoV-2 variant protects hamsters from disease, but not reinfection with B.1.351 SARS-CoV-2 variant.

The circulation of SARS-CoV-2 has resulted in the emergence of variants of concern (VOCs). It is currently unclear whether the previous infection with SARS-CoV-2 provides protection against reinfection with VOCs. Here, we show that low dose aerosol exposure to hCoV-19/human/USA/WA-CDC-WA1/2020 (WA1, lineage A), resulted in a productive mild infection. In contrast, a low dose of SARS-CoV-2 via fomites did not result in productive infection in the majority of exposed hamsters and these animals remained non-seroconverted. After recovery, hamsters were re-exposed to hCoV-19/South African/KRISP-K005325/2020 (VOC B.1.351) via an intranasal challenge. Seroconverted rechallenged animals did not lose weight and shed virus for three days. They had a little infectious virus and no pathology in the lungs. In contrast, shedding, weight loss and extensive pulmonary pathology caused by B.1.351 replication were observed in the non-seroconverted animals. The rechallenged seroconverted animals did not transmit the virus to naïve sentinels via direct contact transmission, in contrast to the non-seroconverted animals. Reinfection with B.1.351 triggered an anamnestic response that boosted not only neutralizing titres against lineage A, but also titres against B.1.351. Our results confirm that aerosol exposure is a more efficient infection route than fomite exposure. Furthermore, initial infection with SARS-CoV-2 lineage A does not prevent heterologous reinfection with B.1.351 but prevents disease and onward transmission. These data suggest that previous SARS-CoV-2 exposure induces partial protective immunity. The reinfection generated a broadly neutralizing humoral response capable of effectively neutralizing B.1.351 while maintaining its ability to neutralize the virus to which the initial response was directed against.

Neutralization diversity of HIV-1 Indian subtype C envelopes obtained from cross sectional and followed up individuals against broadly neutralizing monoclonal antibodies having distinct gp120 specificities.

BACKGROUND: The potential use of the broadly neutralizing monoclonal antibodies (bnAbs) towards prophylaxis and treatment to HIV-1 is currently being explored. While a number of promising bnAbs have been discovered and a few of them have progressed towards clinical development, their extent of neutralization coverage with respect to global HIV-1 variants given the existence of genetically distinct subtypes and recombinants circulating globally is not clearly known. In the present study, we examined the variation in the neutralization susceptibility of pseudoviruses expressing 71 full length primary HIV-1 subtype C envs obtained from limited cross-sectional individuals over different time points against four bnAbs that target gp120 with distinct specificities: VRC01, CAP256-VRC26.25, PGDM1400 and PGT121. RESULTS: We found significant variations in the susceptibility of Indian clade C to these four bnAbs. These variations were found to be distinct to that observed in African subtype C based on the existing datasets and concordant with their sequence diversity. Trend analysis indicated an increasing neutralization resistance observed over time with CAP25-VRC26.25, PGDM1400 and PGT121 when tested on pseudoviruses expressing envs obtained from 1999 to 2016. However, inconsistent trend in neutralization susceptibility was observed, when pseudoviruses expressing envs obtained from three followed up individuals were examined. Finally, through predictive analysis of the 98 Indian subtype C including those assessed in the present study by employing additive model implemented in CombiNAber ( http://www.hiv.lanl.gov ), we observed two possibilities where combinations of three bnAbs (VRC01/CAP56-VRC26.25/PGT121 and PGDM1400/CAP256-VRC26.25/PGT121) could achieve near 100% neutralization coverage. CONCLUSIONS: Our findings not only indicate disparate intra-clade C genetic vis-à-vis neutralization diversities but also warrant the need for more comprehensive study using additional isolates towards comparing inter and intra-clade neutralization diversities which will be necessary for selecting the bnAb combinations suitable for optimal coverage of the region-specific HIV-1 circulating subtypes. Expanding these efforts is imperative for designing efficacious bnAb based intervention strategies for India as well as subtype C in general.

Safety and Immunogenicity of a 4-Component Toxoid-Based Staphylococcus aureus Vaccine in Rhesus Macaques.

Staphylococcus aureus is a leading cause of significant morbidity and mortality and an enormous economic burden to public health worldwide. Infections caused by methicillin-resistant S. aureus (MRSA) pose a major threat as MRSA strains are becoming increasingly prevalent and multi-drug resistant. To this date, vaccines targeting surface-bound antigens demonstrated promising results in preclinical testing but have failed in clinical trials. S. aureus pathogenesis is in large part driven by immune destructive and immune modulating toxins and thus represent promising vaccine targets. Hence, the objective of this study was to evaluate the safety and immunogenicity of a staphylococcal 4-component vaccine targeting secreted bi-component pore-forming toxins (BCPFTs) and superantigens (SAgs) in non-human primates (NHPs). The 4-component vaccine proved to be safe, even when repeated vaccinations were given at a dose that is 5 to 10- fold higher than the proposed human dose. Vaccinated rhesus macaques did not exhibit clinical signs, weight loss, or changes in hematology or serum chemistry parameters related to the administration of the vaccine. No acute, vaccine-related elevation of serum cytokine levels was observed after vaccine administration, confirming the toxoid components lacked superantigenicity. Immunized animals demonstrated high level of toxin-specific total and neutralizing antibodies toward target antigens of the 4-component vaccine as well as cross-neutralizing activity toward staphylococcal BCPFTs and SAgs that are not direct targets of the vaccine. Cross-neutralization was also observed toward the heterologous streptococcal pyogenic exotoxin B. Ex vivo stimulation of PBMCs with individual vaccine components demonstrated an overall increase in several T cell cytokines measured in supernatants. Immunophenotyping of CD4 T cells ex vivo showed an increase in Ag-specific polyfunctional CD4 T cells in response to antigen stimulation. Taken together, we demonstrate that the 4-component vaccine is well-tolerated and immunogenic in NHPs generating both humoral and cellular immune responses. Targeting secreted toxin antigens could be the next-generation vaccine approach for staphylococcal vaccines if also proven to provide efficacy in humans.

A Role for B Cells to Transmit Hepatitis C Virus Infection.

Hepatitis C virus (HCV) is highly variable and transmits through infected blood to establish a chronic liver infection in the majority of patients. Our knowledge on the infectivity of clinical HCV strains is hampered by the lack of in vitro cell culture systems that support efficient viral replication. We and others have reported that HCV can associate with and infect immune cells and may thereby evade host immune surveillance and elimination. To evaluate whether B cells play a role in HCV transmission, we assessed the ability of B cells and sera from recent (<2 years) or chronic (≥ 2 years) HCV patients to infect humanized liver chimeric mice. HCV was transmitted by B cells from chronic infected patients whereas the sera were non-infectious. In contrast, B cells from recently infected patients failed to transmit HCV to the mice, whereas all serum samples were infectious. We observed an association between circulating anti-glycoprotein E1E2 antibodies and B cell HCV transmission. Taken together, our studies provide evidence for HCV transmission by B cells, findings that have clinical implications for prophylactic and therapeutic antibody-based vaccine design.