Determinants of long-lived memory B cells for antiviral immunity

The protection offered by B cell responses after viral infection or vaccination features neutralising antibodies produced by plasmablasts and memory B cells (MBCs) which can provide long lasting immunity against reinfection. Hence understanding the factors associated with MBC persistence is crucial for vaccine design. Murine and human studies have implicated initial antigen affinity, bystander activation, activation-induced cytidine deaminase (AID) expression, and chemokine levels as being associated with persistence of antigen-specific MBCs, but detailed understanding of the genesis of long-lived MBCs in humans is lacking. During the initial phase of COVID-19 pandemic there were multiple studies characterising natural immune responses against SARS-CoV-2 infection, but then the interest shifted towards understanding vaccine-induced immunity. With the spread of variants of concern and breakthrough infections in vaccinees, understanding the longevity of natural immunity is essential to design improved vaccines. The primary objective of this thesis was to develop a highly specific tetramer for flow cytometry-based isolation of SARS-CoV-2 specific MBCs, evaluate their longevity, and identify biomarkers and transcriptomic signatures present early after infection that associate with durable MBCs. The samples used in this thesis were selected from the COVID-19 Outbreak Samples in NSW (COSIN) cohort, an ongoing prospective cohort evaluating the natural history of the SARS-CoV-2 infection in NSW. This thesis observed that at one year post infection, despite declining antibody titers, maintenance of neutralisation breadth and a variant specific protection (45% - 76%) against symptomatic disease. Encouragingly 80% of participants who had SARS-CoV-2 specific MBCs during early convalescence had detectable MBCs at one year. Based on MBC persistence, the cohort was divided into two groups – “Maintained” and “Dropped”. Early antigen specific CD4+ T cell responses was associated with maintenance of antibody neutralisation breadth and MBC durability. Smart-seq2 analysis of the MBCs identified ongoing maturation of the B cell receptor in both grand transcriptomic differences between the two groups. The transcriptomic analysis revealed that an enhanced activation profile of mature, proliferating MBCs was associated with persistence at one year. These findings provide initial identification of biomarkers that can be used to predict the durability of vaccine induced immunity

Zika virus protection by a single low dose nucleoside modified mRNA vaccination

Zika virus (ZIKV) has recently emerged as an explosive pandemic associated with severe neuropathology in newborns and adults1. There are no ZIKV-specific treatments or preventatives; thus, development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins2,3. Here, we demonstrate that a single low-dose intradermal immunization with lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope (prM-E) glycoproteins of a 2013 ZIKV outbreak strain elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 μg of nucleoside-modified ZIKV mRNA-LNPs protected mice from ZIKV challenges at 2 weeks or 5 months post-vaccination, and a single dose of 50 μg was sufficient to protect non-human primates from a challenge at 5 weeks post-vaccination. These data demonstrate that nucleoside-modified mRNA-LNPs elicit rapid and durable protective immunity and thus represent a new and promising vaccine candidate for the global fight against ZIKV

Pentavalent HIV-1 vaccine protects against simian-human immunodeficiency virus challenge

The RV144 Thai trial HIV-1 vaccine of recombinant poxvirus (ALVAC) and recombinant HIV-1 gp120 subtype B/subtype E (B/E) proteins demonstrated 31% vaccine efficacy. Here we design an ALVAC/Pentavalent B/E/E/E/E vaccine to increase the diversity of gp120 motifs in the immunogen to elicit a broader antibody response and enhance protection. We find that immunization of rhesus macaques with the pentavalent vaccine results in protection of 55% of pentavalent-vaccine-immunized macaques from simian–human immunodeficiency virus (SHIV) challenge. Systems serology of the antibody responses identifies plasma antibody binding to HIV-infected cells, peak ADCC antibody titres, NK cell-mediated ADCC and antibody-mediated activation of MIP-1β in NK cells as the four immunological parameters that best predict decreased infection risk that are improved by the pentavalent vaccine. Thus inclusion of additional gp120 immunogens to a pox-prime/protein boost regimen can augment antibody responses and enhance protection from a SHIV challenge in rhesus macaques

Vaccination Reduces Simian-Human Immunodeficiency Virus Sequence Reversion through Enhanced Viral Control▿

It has been suggested that vaccination prior to infection may direct the mutational evolution of human immunodeficiency virus type 1 (HIV-1) to a less fit virus, resulting in an attenuated course of disease. The present study was initiated to explore whether prior immunization might prevent the reversion of the virus to the wild-type form. Mamu-A*01 monkeys were vaccinated to generate a cytotoxic T-lymphocyte response to the immunodominant Gag p11C epitope and were then challenged with a cloned pathogenic CXCR4-tropic simian-human immunodeficiency virus (SHIV) expressing a mutant Gag p11C sequence (Δp11C SHIV). The epitopic and extraepitopic compensatory mutations introduced into gag of Δp11C SHIV resulted in attenuated replicative capacity and eventual reversions to the wild-type Gag p11C sequence in naïve rhesus monkeys. However, in vaccinated rhesus monkeys, no reversions of the challenge virus were observed, an effect that may have been a consequence of significantly decreased viral replication rather than a redirection of the mutational evolution of the virus. These findings highlight the multifactorial pressures that affect the evolution of primate immunodeficiency viruses

Cross-reactive potential of human T-lymphocyte responses in HIV-1 infection

An effective HIV-1 vaccine should elicit sufficient breadth of immune recognition to protect against the genetically diverse forms of the circulating virus. Evaluation of the breadth and magnitude of cellular immune responses to epitope variants is important for HIV-1 vaccine assessment. We compared HIV-1 Gag-specific T-lymphocyte responses in 20 HIV-1-infected individuals representing two different HIV-1 subtypes, B and C. By assessing T lymphocyte responses with peptides based on natural HIV-1 variants, we found evidence for limited cross-reactivity and significantly enhanced within-clade responses among clade B-infected subjects, and not among clade C-infected subjects

Evaluation of a prime-boost vaccine schedule with distinct adenovirus vectors against malaria in rhesus monkeys

A vaccine that elicits both specific antibodies and IFN-gamma-producing T cells is required to protect against pre-erythrocytic malaria. Among the most promising approaches to induce such complex immunity are heterologous prime-boost vaccination regiments, in particular ones containing liver viral vector. We have demonstrated previously that adenovectors serotype 35 (Ads35) encoding the circumsporozoite (CS) antigen or liver-stage antigen-1 (LSA-1) are highly effective in improving the T-cell responses induced by immunizations with protein-based vaccines in a heterologous prime-boost schedule. Here we evaluated the potential of a heterologous prime-boost vaccination that combines the Ad35.CS vector with the serologically distinct adenovector AD5.Cs in rhesus macaques, after establishing the potency in mice. We show that the heterologous Ad35.CS/Ad5.Cs prime-boost regimen elicitis both antibody responses and robust IFN-gamma-prodcuting CD8(+) T-cell responses against the CS antigen. Analysis of the quality of the antibody responses in rhesus macaques, using indirect immunofluorescence assay (IFA) with Plasmodium falciparum-coated slides, demonstrated that this heterologous prime-boost regimen elicits a high titer of antibodies that are able to bind to P. falciparum sporozites. Level of the IFA response was superior to the response measured with sera of an adult human population living in endemic malaria region. In conclusion, the combination of Ad35.CS, a vaccine based on a rare serotype adenovirus, with Ad5.CS or possibly another adenovector of a distinct serotype, induces a complex immune response that is required for protection against malaria, and is thus a highly promising approach for pediatric vaccination. (C) 2009 Elseiver Ltd. All rights reserve

Recombinant Mycobacterium bovis BCG Prime-Recombinant Adenovirus Boost Vaccination in Rhesus Monkeys Elicits Robust Polyfunctional Simian Immunodeficiency Virus-Specific T-Cell Responses▿

While mycobacteria have been proposed as vaccine vectors because of their persistence and safety, little has been done systematically to optimize their immunogenicity in nonhuman primates. We successfully generated recombinant Mycobacterium bovis BCG (rBCG) expressing simian immunodeficiency virus (SIV) Gag and Pol as multigenic, nonintegrating vectors, but rBCG-expressing SIV Env was unstable. A dose and route determination study in rhesus monkeys revealed that intramuscular administration of rBCG was associated with local reactogenicity, whereas intravenous and intradermal administration of 106 to 108 CFU of rBCG was well tolerated. After single or repeat rBCG inoculations, monkeys developed high-frequency gamma interferon enzyme-linked immunospot responses against BCG purified protein derivative. However, the same animals developed only modest SIV-specific CD8+ T-cell responses. Nevertheless, high-frequency SIV-specific cellular responses were observed in the rBCG-primed monkeys after boosting with recombinant adenovirus 5 (rAd5) expressing the SIV antigens. These cellular responses were of greater magnitude and more persistent than those generated after vaccination with rAd5 alone. The vaccine-elicited cellular responses were predominantly polyfunctional CD8+ T cells. These findings support the further exploration of mycobacteria as priming vaccine vectors

Breadth of cellular and humoral immune responses elicited in rhesus monkeys by multi-valent mosaic and consensus immunogens

To create an HIV-1 vaccine that generates sufficient breadth of immune recognition to protect against the genetically diverse forms of the circulating virus, we have been exploring vaccines based on consensus and mosaic protein designs. Increasing the valency of a mosaic immunogen cocktail increases epitope coverage but with diminishing returns, as increasingly rare epitopes are incorporated into the mosaic proteins. In this study we compared the immunogenicity of 2-valent and 3-valent HIV-1 envelope mosaic immunogens in rhesus monkeys. Immunizations with the 3-valent mosaic immunogens resulted in a modest increase in the breadth of vaccine-elicited T lymphocyte responses compared to the 2-valent mosaic immunogens. However, the 3-valent mosaic immunogens elicited significantly higher neutralizing responses to Tier 1 viruses than the 2-valent mosaic immunogens. These findings underscore the potential utility of polyvalent mosaic immunogens for eliciting both cellular and humoral immune responses to HIV-1