Vaccination with antigenically complex hemagglutinin mixtures confers broad protection from influenza disease.

Current seasonal influenza virus vaccines induce responses primarily against immunodominant but highly plastic epitopes in the globular head of the hemagglutinin (HA) glycoprotein. Because of viral antigenic drift at these sites, vaccines need to be updated and readministered annually. To increase the breadth of influenza vaccine-mediated protection, we developed an antigenically complex mixture of recombinant HAs designed to redirect immune responses to more conserved domains of the protein. Vaccine-induced antibodies were disproportionally redistributed to the more conserved stalk of the HA without hindering, and in some cases improving, antibody responses against the head domain. These improved responses led to increased protection against homologous and heterologous viral challenges in both mice and ferrets compared with conventional vaccine approaches. Thus, antigenically complex protein mixtures can at least partially overcome HA head domain antigenic immunodominance and may represent a step toward a more universal influenza vaccine.

Multivariate analysis of FcR-mediated NK cell functions identifies unique clustering among humans and rhesus macaques.

Rhesus macaques (RMs) are a common pre-clinical model used to test HIV vaccine efficacy and passive immunization strategies. Yet, it remains unclear to what extent the Fc-Fc receptor (FcR) interactions impacting antiviral activities of antibodies in RMs recapitulate those in humans. Here, we evaluated the FcR-related functionality of natural killer cells (NKs) from peripheral blood of uninfected humans and RMs to identify intra- and inter-species variation. NKs were screened for FcγRIIIa (human) and FcγRIII (RM) genotypes (FcγRIII(a)), receptor signaling, and antibody-dependent cellular cytotoxicity (ADCC), the latter mediated by a cocktail of monoclonal IgG1 antibodies with human or RM Fc. FcγRIII(a) genetic polymorphisms alone did not explain differences in NK effector functionality in either species cohort. Using the same parameters, hierarchical clustering separated each species into two clusters. Importantly, in principal components analyses, ADCC magnitude, NK contribution to ADCC, FcγRIII(a) cell-surface expression, and frequency of phosphorylated CD3ζ NK cells all contributed similarly to the first principal component within each species, demonstrating the importance of measuring multiple facets of NK cell function. Although ADCC potency was similar between species, we detected significant differences in frequencies of NK cells and pCD3ζ+ cells, level of cell-surface FcγRIII(a) expression, and NK-mediated ADCC (P<0.001), indicating that a combination of Fc-FcR parameters contribute to overall inter-species functional differences. These data strongly support the importance of multi-parameter analyses of Fc-FcR NK-mediated functions when evaluating efficacy of passive and active immunizations in pre- and clinical trials and identifying correlates of protection. The results also suggest that pre-screening animals for multiple FcR-mediated NK function would ensure even distribution of animals among treatment groups in future preclinical trials.

Viral vector delivered immunogen focuses HIV-1 antibody specificity and increases durability of the circulating antibody recall response.

The modestly efficacious HIV-1 vaccine regimen (RV144) conferred 31% vaccine efficacy at 3 years following the four-shot immunization series, coupled with rapid waning of putative immune correlates of decreased infection risk. New strategies to increase magnitude and durability of protective immunity are critically needed. The RV305 HIV-1 clinical trial evaluated the immunological impact of a follow-up boost of HIV-1-uninfected RV144 recipients after 6-8 years with RV144 immunogens (ALVAC-HIV alone, AIDSVAX B/E gp120 alone, or ALVAC-HIV + AIDSVAX B/E gp120). Previous reports demonstrated that this regimen elicited higher binding, antibody Fc function, and cellular responses than the primary RV144 regimen. However, the impact of the canarypox viral vector in driving antibody specificity, breadth, durability and function is unknown. We performed a follow-up analysis of humoral responses elicited in RV305 to determine the impact of the different booster immunogens on HIV-1 epitope specificity, antibody subclass, isotype, and Fc effector functions. Importantly, we observed that the ALVAC vaccine component directly contributed to improved breadth, function, and durability of vaccine-elicited antibody responses. Extended boosts in RV305 increased circulating antibody concentration and coverage of heterologous HIV-1 strains by V1V2-specific antibodies above estimated protective levels observed in RV144. Antibody Fc effector functions, specifically antibody-dependent cellular cytotoxicity and phagocytosis, were boosted to higher levels than was achieved in RV144. V1V2 Env IgG3, a correlate of lower HIV-1 risk, was not increased; plasma Env IgA (specifically IgA1), a correlate of increased HIV-1 risk, was elevated. The quality of the circulating polyclonal antibody response changed with each booster immunization. Remarkably, the ALVAC-HIV booster immunogen induced antibody responses post-second boost, indicating that the viral vector immunogen can be utilized to selectively enhance immune correlates of decreased HIV-1 risk. These results reveal a complex dynamic of HIV-1 immunity post-vaccination that may require careful balancing to achieve protective immunity in the vaccinated population. Trial registration: RV305 clinical trial (ClinicalTrials.gov number, NCT01435135). ClinicalTrials.gov Identifier: NCT00223080.

A tool for evaluating heterogeneity in avidity of polyclonal antibodies.

Diversity in specificity of polyclonal antibody (pAb) responses is extensively investigated in vaccine efficacy or immunological evaluations, but the heterogeneity in antibody avidity is rarely probed as convenient tools are lacking. Here we have developed a polyclonal antibodies avidity resolution tool (PAART) for use with label-free techniques, such as surface plasmon resonance and biolayer interferometry, that can monitor pAb-antigen interactions in real time to measure dissociation rate constant (kd ) for defining avidity. PAART utilizes a sum of exponentials model to fit the dissociation time-courses of pAb-antigens interactions and resolve multiple kd contributing to the overall dissociation. Each kd value of pAb dissociation resolved by PAART corresponds to a group of antibodies with similar avidity. PAART is designed to identify the minimum number of exponentials required to explain the dissociation course and guards against overfitting of data by parsimony selection of best model using Akaike information criterion. Validation of PAART was performed using binary mixtures of monoclonal antibodies of same specificity but differing in kd of the interaction with their epitope. We applied PAART to examine the heterogeneity in avidities of pAb from malaria and typhoid vaccinees, and individuals living with HIV-1 that naturally control the viral load. In many cases, two to three kd were dissected indicating the heterogeneity of pAb avidities. We showcase examples of affinity maturation of vaccine induced pAb responses at component level and enhanced resolution of heterogeneity in avidity when antigen-binding fragments (Fab) are used instead of polyclonal IgG antibodies. The utility of PAART can be manifold in examining circulating pAb characteristics and could inform vaccine strategies aimed to guide the host humoral immune response.

Impact of adjuvants on the biophysical and functional characteristics of HIV vaccine-elicited antibodies in humans.

Adjuvants can alter the magnitude, characteristics, and persistence of the humoral response to protein vaccination. HIV vaccination might benefit from tailored adjuvant choice as raising a durable and protective response to vaccination has been exceptionally challenging. Analysis of trials of partially effective HIV vaccines have identified features of the immune response that correlate with decreased risk, including high titers of V1V2-binding IgG and IgG3 responses with low titers of V1V2-binding IgA responses and enhanced Fc effector functions, notably antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). However, there has been limited opportunity to compare the effect of different adjuvants on these activities in humans. Here, samples from the AVEG015 study, a phase 1 trial in which participants (n = 112) were immunized with gp120SF-2 and one of six different adjuvants or combinations thereof were assessed for antibody titer, biophysical features, and diverse effector functions. Three adjuvants, MF59 + MTP-PE, SAF/2, and SAF/2 + MDP, increased the peak magnitude and durability of antigen-specific IgG3, IgA, FcγR-binding responses and ADCP activity, as compared to alum. While multiple adjuvants increased the titer of IgG, IgG3, and IgA responses, none consistently altered the balance of IgG to IgA or IgG3 to IgA. Linear regression analysis identified biophysical features including gp120-specific IgG and FcγR-binding responses that could predict functional activity, and network analysis identified coordinated aspects of the humoral response. These analyses reveal the ability of adjuvants to drive the character and function of the humoral response despite limitations of small sample size and immune variability in this human clinical trial.

Subclass and avidity of circumsporozoite protein specific antibodies associate with protection status against malaria infection.

RTS,S/AS01 is an advanced pre-erythrocytic malaria vaccine candidate with demonstrated vaccine efficacy up to 86.7% in controlled human malaria infection (CHMI) studies; however, reproducible immune correlates of protection (CoP) are elusive. To identify candidates of humoral correlates of vaccine mediated protection, we measured antibody magnitude, subclass, and avidity for Plasmodium falciparum (Pf) circumsporozoite protein (CSP) by multiplex assays in two CHMI studies with varying RTS,S/AS01B vaccine dose and timing regimens. Central repeat (NANP6) IgG1 magnitude correlated best with protection status in univariate analyses and was the most predictive for protection in a multivariate model. NANP6 IgG3 magnitude, CSP IgG1 magnitude, and total serum antibody dissociation phase area-under-the-curve for NANP6, CSP, NPNA3, and N-interface binding were also associated with protection status in the regimen adjusted univariate analysis. Identification of multiple immune response features that associate with protection status, such as antibody subclasses, fine specificity and avidity reported here may accelerate development of highly efficacious vaccines against P. falciparum.

Comprehensive Data Integration Approach to Assess Immune Responses and Correlates of RTS,S/AS01-Mediated Protection From Malaria Infection in Controlled Human Malaria Infection Trials.

RTS,S/AS01 (GSK) is the world's first malaria vaccine. However, despite initial efficacy of almost 70% over the first 6 months of follow-up, efficacy waned over time. A deeper understanding of the immune features that contribute to RTS,S/AS01-mediated protection could be beneficial for further vaccine development. In two recent controlled human malaria infection (CHMI) trials of the RTS,S/AS01 vaccine in malaria-naïve adults, MAL068 and MAL071, vaccine efficacy against patent parasitemia ranged from 44% to 87% across studies and arms (each study included a standard RTS,S/AS01 arm with three vaccine doses delivered in four-week-intervals, as well as an alternative arm with a modified version of this regimen). In each trial, RTS,S/AS01 immunogenicity was interrogated using a broad range of immunological assays, assessing cellular and humoral immune parameters as well as gene expression. Here, we used a predictive modeling framework to identify immune biomarkers measured at day-of-challenge that could predict sterile protection against malaria infection. Using cross-validation on MAL068 data (either the standard RTS,S/AS01 arm alone, or across both the standard RTS,S/AS01 arm and the alternative arm), top-performing univariate models identified variables related to Fc effector functions and titer of antibodies that bind to the central repeat region (NANP6) of CSP as the most predictive variables; all NANP6-related variables consistently associated with protection. In cross-study prediction analyses of MAL071 outcomes (the standard RTS,S/AS01 arm), top-performing univariate models again identified variables related to Fc effector functions of NANP6-targeting antibodies as highly predictive. We found little benefit-with this dataset-in terms of improved prediction accuracy in bivariate models vs. univariate models. These findings await validation in children living in malaria-endemic regions, and in vaccinees administered a fourth RTS,S/AS01 dose. Our findings support a "quality as well as quantity" hypothesis for RTS,S/AS01-elicited antibodies against NANP6, implying that malaria vaccine clinical trials should assess both titer and Fc effector functions of anti-NANP6 antibodies.

Functional Homology for Antibody-Dependent Phagocytosis Across Humans and Rhesus Macaques.

Analyses of human clinical HIV-1 vaccine trials and preclinical vaccine studies performed in rhesus macaque (RM) models have identified associations between non-neutralizing Fc Receptor (FcR)-dependent antibody effector functions and reduced risk of infection. Specifically, antibody-dependent phagocytosis (ADP) has emerged as a common correlate of reduced infection risk in multiple RM studies and the human HVTN505 trial. This recurrent finding suggests that antibody responses with the capability to mediate ADP are most likely a desirable component of vaccine responses aimed at protecting against HIV-1 acquisition. As use of RM models is essential for development of the next generation of candidate HIV-1 vaccines, there is a need to determine how effectively ADP activity observed in RMs translates to activity in humans. In this study we compared ADP activity of human and RM monocytes and polymorphonuclear leukocytes (PMN) to bridge this gap in knowledge. We observed considerable variability in the magnitude of monocyte and PMN ADP activity across individual humans and RM that was not dependent on FcR alleles, and only modestly impacted by cell-surface levels of FcRs. Importantly, we found that for both human and RM phagocytes, ADP activity of antibodies targeting the CD4 binding site was greatest when mediated by human IgG3, followed by RM and human IgG1. These results demonstrate that there is functional homology between antibody and FcRs from these two species for ADP. We also used novel RM IgG1 monoclonal antibodies engineered with elongated hinge regions to show that hinge elongation augments RM ADP activity. The RM IgGs with engineered hinge regions can achieve ADP activity comparable to that observed with human IgG3. These novel modified antibodies will have utility in passive immunization studies aimed at defining the role of IgG3 and ADP in protection from virus challenge or control of disease in RM models. Our results contribute to a better translation of human and macaque antibody and FcR biology, and may help to improve testing accuracy and evaluations of future active and passive prevention strategies.

Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses.

Betacoronaviruses caused the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome, as well as the current pandemic of SARS coronavirus 2 (SARS-CoV-2)1-4. Vaccines that elicit protective immunity against SARS-CoV-2 and betacoronaviruses that circulate in animals have the potential to prevent future pandemics. Here we show that the immunization of macaques with nanoparticles conjugated with the receptor-binding domain of SARS-CoV-2, and adjuvanted with 3M-052 and alum, elicits cross-neutralizing antibody responses against bat coronaviruses, SARS-CoV and SARS-CoV-2 (including the B.1.1.7, P.1 and B.1.351 variants). Vaccination of macaques with these nanoparticles resulted in a 50% inhibitory reciprocal serum dilution (ID50) neutralization titre of 47,216 (geometric mean) for SARS-CoV-2, as well as in protection against SARS-CoV-2 in the upper and lower respiratory tracts. Nucleoside-modified mRNAs that encode a stabilized transmembrane spike or monomeric receptor-binding domain also induced cross-neutralizing antibody responses against SARS-CoV and bat coronaviruses, albeit at lower titres than achieved with the nanoparticles. These results demonstrate that current mRNA-based vaccines may provide some protection from future outbreaks of zoonotic betacoronaviruses, and provide a multimeric protein platform for the further development of vaccines against multiple (or all) betacoronaviruses.

SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys.

Betacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.

SIMON: Open-Source Knowledge Discovery Platform.

Data analysis and knowledge discovery has become more and more important in biology and medicine with the increasing complexity of biological datasets, but the necessarily sophisticated programming skills and in-depth understanding of algorithms needed pose barriers to most biologists and clinicians to perform such research. We have developed a modular open-source software, SIMON, to facilitate the application of 180+ state-of-the-art machine-learning algorithms to high-dimensional biomedical data. With an easy-to-use graphical user interface, standardized pipelines, and automated approach for machine learning and other statistical analysis methods, SIMON helps to identify optimal algorithms and provides a resource that empowers non-technical and technical researchers to identify crucial patterns in biomedical data.

Polyclonal Broadly Neutralizing Antibody Activity Characterized by CD4 Binding Site and V3-Glycan Antibodies in a Subset of HIV-1 Virus Controllers.

Broadly neutralizing antibodies (bNAbs), known to mediate immune control of HIV-1 infection, only develop in a small subset of HIV-1 infected individuals. Despite being traditionally associated with patients with high viral loads, bNAbs have also been observed in therapy naïve HIV-1+ patients naturally controlling virus replication [Virus Controllers (VCs)]. Thus, dissecting the bNAb response in VCs will provide key information about what constitutes an effective humoral response to natural HIV-1 infection. In this study, we identified a polyclonal bNAb response to natural HIV-1 infection targeting CD4 binding site (CD4bs), V3-glycan, gp120-gp41 interface and membrane-proximal external region (MPER) epitopes on the HIV-1 envelope (Env). The polyclonal antiviral antibody (Ab) response also included antibody-dependent cellular phagocytosis of clade AE, B and C viruses, consistent with both the Fv and Fc domain contributing to function. Sequence analysis of envs from one of the VCs revealed features consistent with potential immune pressure and virus escape from V3-glycan targeting bNAbs. Epitope mapping of the polyclonal bNAb response in VCs with bNAb activity highlighted the presence of gp120-gp41 interface and CD4bs antibody classes with similar binding profiles to known potent bNAbs. Thus, these findings reveal the induction of a broad and polyfunctional humoral response in VCs in response to natural HIV-1 infection.

Vi-specific serological correlates of protection for typhoid fever.

Typhoid Vi vaccines have been shown to be efficacious in children living in endemic regions; however, a widely accepted correlate of protection remains to be established. We applied a systems serology approach to identify Vi-specific serological correlates of protection using samples obtained from participants enrolled in an experimental controlled human infection study. Participants were vaccinated with Vi-tetanus toxoid conjugate (Vi-TT) or unconjugated Vi-polysaccharide (Vi-PS) vaccines and were subsequently challenged with Salmonella Typhi bacteria. Multivariate analyses identified distinct protective signatures for Vi-TT and Vi-PS vaccines in addition to shared features that predicted protection across both groups. Vi IgA quantity and avidity correlated with protection from S. Typhi infection, whereas higher fold increases in Vi IgG responses were associated with reduced disease severity. Targeted antibody-mediated functional responses, particularly neutrophil phagocytosis, were also identified as important components of the protective signature. These humoral markers could be used to evaluate and develop efficacious Vi-conjugate vaccines and assist with accelerating vaccine availability to typhoid-endemic regions.

HIV-1 Vaccine Sequences Impact V1V2 Antibody Responses: A Comparison of Two Poxvirus Prime gp120 Boost Vaccine Regimens.

In the RV144 trial, vaccine-induced V1V2 IgG correlated with decreased HIV-1 risk. We investigated circulating antibody specificities in two phase 1 poxvirus prime-protein boost clinical trials conducted in South Africa: HVTN 097 (subtype B/E) and HVTN 100 (subtype C). With cross-subtype peptide microarrays and multiplex binding assays, we probed the magnitude and breadth of circulating antibody responses to linear variable loop 2 (V2) and conformational V1V2 specificities. Antibodies targeting the linear V2 epitope, a correlate of decreased HIV-1 risk in RV144, were elicited up to 100% and 61% in HVTN 097 and HVTN 100, respectively. Despite higher magnitude of envelope-specific responses in HVTN 100 compared to HVTN 097 (p's < 0.001), the magnitude and positivity for V2 linear epitope and V1V2 proteins were significantly lower in HVTN 100 compared to HVTN 097. Meanwhile, responses to other major linear epitopes including the variable 3 (V3) and constant 5 (C5) epitopes were higher in HVTN 100 compared to HVTN 097. Our data reveal substantial differences in the circulating antibody specificities induced by vaccination in these two canarypox prime-protein boost trials. Our findings suggest that the choice of viral sequences in prime-boost vaccine regimens, and potentially adjuvants and immunogen dose, influence the elicitation of V2-specific antibodies.

IgA and IgG1 Specific to Vi Polysaccharide of Salmonella Typhi Correlate With Protection Status in a Typhoid Fever Controlled Human Infection Model.

Vaccination against Salmonella Typhi using the Vi capsular polysaccharide, a T-cell independent antigen, can protect from the development of typhoid fever. This implies that antibodies to Vi alone can protect in the absence of a T cell-mediated immune response; however, protective Vi antibodies have not been well-characterized. We hypothesized that variability in the biophysical properties of vaccine-elicited antibodies, including subclass distribution and avidity, may impact protective outcomes. To interrogate the relationship between antibody properties and protection against typhoid fever, we analyzed humoral responses from participants in a vaccine efficacy (VE) trial using a controlled human infection model (CHIM) who received either a purified Vi polysaccharide (Vi-PS) or Vi tetanus toxoid conjugate (Vi-TT) vaccine followed by oral challenge with live S. Typhi. We determined the avidity, overall magnitude, and vaccine-induced fold-change in magnitude from before immunization to day of challenge of Vi IgA and IgG subclass antibodies. Amongst those who received the Vi-PS vaccine, Vi IgA magnitude (FDR p = 0.01) and fold-change (FDR p = 0.02) were significantly higher in protected individuals compared with those individuals who developed disease ("diagnosed"). In the Vi-TT vaccine group, the responses of protected individuals had higher fold-change in Vi IgA (FDR p = 0.06) and higher Vi IgG1 avidity (FDR p = 0.058) than the diagnosed Vi-TT vaccinees, though these findings were not significant at p < 0.05. Overall, protective antibody signatures differed between the Vi-PS and Vi-TT vaccines, thus, we conclude that although the Vi-PS and Vi-TT vaccines were observed to have similar efficacies, these vaccines may protect through different mechanisms. These data will inform studies on mechanisms of protection against typhoid fever, including identification of antibody effector functions, as well as informing future vaccination strategies.