Christine A. Bricault

Vaccine protection against Zika virus from Brazil

Zika virus (ZIKV) is a flavivirus that is responsible for the current epidemic in Brazil and the Americas. ZIKV has been causally associated with fetal microcephaly, intrauterine growth restriction, and other birth defects in both humans and mice. The rapid development of a safe and effective ZIKV vaccine is a global health priority, but very little is currently known about ZIKV immunology and mechanisms of immune protection. Here we show that a single immunization with a plasmid DNA vaccine or a purified inactivated virus vaccine provides complete protection in susceptible mice against challenge with a strain of ZIKV involved in the outbreak in northeast Brazil. This ZIKV strain has recently been shown to cross the placenta and to induce fetal microcephaly and other congenital malformations in mice. We produced DNA vaccines expressing ZIKV pre-membrane and envelope (prM-Env), as well as a series of deletion mutants. The prM-Env DNA vaccine, but not the deletion mutants, afforded complete protection against ZIKV, as measured by absence of detectable viraemia following challenge, and protective efficacy correlated with Env-specific antibody titers. Adoptive transfer of purified IgG from vaccinated mice conferred passive protection, and depletion of CD4 and CD8 T lymphocytes in vaccinated mice did not abrogate this protection. These data demonstrate that protection against ZIKV challenge can be achieved by single-shot subunit and inactivated virus vaccines in mice and that Env-specific antibody titers represent key immunologic correlates of protection. Our findings suggest that the development of a ZIKV vaccine for humans is likely to be achievable.

Protective efficacy of multiple vaccine platforms against Zika virus challenge in rhesus monkeys

Zika virus (ZIKV) is responsible for a major ongoing epidemic in the Americas and has been causally associated with fetal microcephaly. The development of a safe and effective ZIKV vaccine is therefore an urgent global health priority. Here we demonstrate that three different vaccine platforms protect against ZIKV challenge in rhesus monkeys. A purified inactivated virus vaccine induced ZIKV-specific neutralizing antibodies and completely protected monkeys against ZIKV strains from both Brazil and Puerto Rico. Purified immunoglobulin from vaccinated monkeys also conferred passive protection in adoptive transfer studies. A plasmid DNA vaccine and a single-shot recombinant rhesus adenovirus serotype 52 vector vaccine, both expressing ZIKV premembrane and envelope, also elicited neutralizing antibodies and completely protected monkeys against ZIKV challenge. These data support the rapid clinical development of ZIKV vaccines for humans.

Preliminary aggregate safety and immunogenicity results from three trials of a purified inactivated Zika virus vaccine candidate: phase 1, randomised, double-blind, placebo-controlled clinical trials

BACKGROUND A safe, effective, and rapidly scalable vaccine against Zika virus infection is needed. We developed a purified formalin-inactivated Zika virus vaccine (ZPIV) candidate that showed protection in mice and non-human primates against viraemia after Zika virus challenge. Here we present the preliminary results in human beings. METHODS We did three phase 1, placebo-controlled, double-blind trials of ZPIV with aluminium hydroxide adjuvant. In all three studies, healthy adults were randomly assigned by a computer-generated list to receive 5 μg ZPIV or saline placebo, in a ratio of 4:1 at Walter Reed Army Institute of Research, Silver Spring, MD, USA, or of 5:1 at Saint Louis University, Saint Louis, MO, USA, and Beth Israel Deaconess Medical Center, Boston, MA, USA. Vaccinations were given intramuscularly on days 1 and 29. The primary objective was safety and immunogenicity of the ZPIV candidate. We recorded adverse events and Zika virus envelope microneutralisation titres up to day 57. These trials are registered at ClinicalTrials.gov, numbers NCT02963909, NCT02952833, and NCT02937233. FINDINGS We enrolled 68 participants between Nov 7, 2016, and Jan 25, 2017. One was excluded and 67 participants received two injections of Zika vaccine (n=55) or placebo (n=12). The vaccine caused only mild to moderate adverse events. The most frequent local effects were pain (n=40 [60%]) or tenderness (n=32 [47%]) at the injection site, and the most frequent systemic reactogenic events were fatigue (29 [43%]), headache (26 [39%]), and malaise (15 [22%]). By day 57, 52 (92%) of vaccine recipients had seroconverted (microneutralisation titre ≥1:10), with peak geometric mean titres seen at day 43 and exceeding protective thresholds seen in animal studies. INTERPRETATION The ZPIV candidate was well tolerated and elicited robust neutralising antibody titres in healthy adults. FUNDING Departments of the Army and Defense and National Institute of Allergy and Infectious Diseases.

A Multivalent Clade C HIV-1 Env Trimer Cocktail Elicits a Higher Magnitude of Neutralizing Antibodies than Any Individual Component

ABSTRACT The sequence diversity of human immunodeficiency virus type 1 (HIV-1) presents a formidable challenge to the generation of an HIV-1 vaccine. One strategy to address such sequence diversity and to improve the magnitude of neutralizing antibodies (NAbs) is to utilize multivalent mixtures of HIV-1 envelope (Env) immunogens. Here we report the generation and characterization of three novel, acute clade C HIV-1 Env gp140 trimers (459C, 405C, and 939C), each with unique antigenic properties. Among the single trimers tested, 459C elicited the most potent NAb responses in vaccinated guinea pigs. We evaluated the immunogenicity of various mixtures of clade C Env trimers and found that a quadrivalent cocktail of clade C trimers elicited a greater magnitude of NAbs against a panel of tier 1A and 1B viruses than any single clade C trimer alone, demonstrating that the mixture had an advantage over all individual components of the cocktail. These data suggest that vaccination with a mixture of clade C Env trimers represents a promising strategy to augment vaccine-elicited NAb responses. IMPORTANCE It is currently not known how to generate potent NAbs to the diverse circulating HIV-1 Envs by vaccination. One strategy to address this diversity is to utilize mixtures of different soluble HIV-1 envelope proteins. In this study, we generated and characterized three distinct, novel, acute clade C soluble trimers. We vaccinated guinea pigs with single trimers as well as mixtures of trimers, and we found that a mixture of four trimers elicited a greater magnitude of NAbs than any single trimer within the mixture. The results of this study suggest that further development of Env trimer cocktails is warranted.

Characterization and Immunogenicity of a Novel Mosaic M HIV-1 gp140 Trimer

ABSTRACT The extraordinary diversity of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein poses a major challenge for the development of an HIV-1 vaccine. One strategy to circumvent this problem utilizes bioinformatically optimized mosaic antigens. However, mosaic Env proteins expressed as trimers have not been previously evaluated for their stability, antigenicity, and immunogenicity. Here, we report the production and characterization of a stable HIV-1 mosaic M gp140 Env trimer. The mosaic M trimer bound CD4 as well as multiple broadly neutralizing monoclonal antibodies, and biophysical characterization suggested substantial stability. The mosaic M trimer elicited higher neutralizing antibody (nAb) titers against clade B viruses than a previously described clade C (C97ZA.012) gp140 trimer in guinea pigs, whereas the clade C trimer elicited higher nAb titers than the mosaic M trimer against clade A and C viruses. A mixture of the clade C and mosaic M trimers elicited nAb responses that were comparable to the better component of the mixture for each virus tested. These data suggest that combinations of relatively small numbers of immunologically complementary Env trimers may improve nAb responses. IMPORTANCE The development of an HIV-1 vaccine remains a formidable challenge due to multiple circulating strains of HIV-1 worldwide. This study describes a candidate HIV-1 Env protein vaccine whose sequence has been designed by computational methods to address HIV-1 diversity. The characteristics and immunogenicity of this Env protein, both alone and mixed together with a clade C Env protein vaccine, are described.

Construction and Evaluation of Novel Rhesus Monkey Adenovirus Vaccine Vectors

ABSTRACT Adenovirus vectors are widely used as vaccine candidates for a variety of pathogens, including HIV-1. To date, human and chimpanzee adenoviruses have been explored in detail as vaccine vectors. The phylogeny of human and chimpanzee adenoviruses is overlapping, and preexisting humoral and cellular immunity to both are exhibited in human populations worldwide. More distantly related adenoviruses may therefore offer advantages as vaccine vectors. Here we describe the primary isolation and vectorization of three novel adenoviruses from rhesus monkeys. The seroprevalence of these novel rhesus monkey adenovirus vectors was extremely low in sub-Saharan Africa human populations, and these vectors proved to have immunogenicity comparable to that of human and chimpanzee adenovirus vaccine vectors in mice. These rhesus monkey adenoviruses phylogenetically clustered with the poorly described adenovirus species G and robustly stimulated innate immune responses. These novel adenoviruses represent a new class of candidate vaccine vectors. IMPORTANCE Although there have been substantial efforts in the development of vaccine vectors from human and chimpanzee adenoviruses, far less is known about rhesus monkey adenoviruses. In this report, we describe the isolation and vectorization of three novel rhesus monkey adenoviruses. These vectors exhibit virologic and immunologic characteristics that make them attractive as potential candidate vaccine vectors for both HIV-1 and other pathogens.

Alteration of intersubunit acid-base pair interactions at the quasi-threefold axis of symmetry of Cucumber mosaic virus disrupts aphid vector transmission.

In the atomic model of Cucumber mosaic virus (CMV), six amino acid residues form stabilizing salt bridges between subunits of the asymmetric unit at the quasi-threefold axis of symmetry. To evaluate the effects of these positions on virion stability and aphid vector transmissibility, six charged amino acid residues were individually mutated to alanine. All of the six engineered viruses were viable and exhibited near wild type levels of virion stability in the presence of urea. Aphid vector transmissibility was nearly or completely eliminated in the case of four of the mutants; two mutants demonstrated intermediate aphid transmissibility. For the majority of the engineered mutants, second-site mutations were observed following aphid transmission and/or mechanical passaging, and one restored transmission rates to that of the wild type. CMV capsids tolerate disruption of acid-base pairing interactions at the quasi-threefold axis of symmetry, but these interactions are essential for maintaining aphid vector transmissibility.

Adenovirus serotype 5 vaccine vectors trigger IL-27–dependent inhibitory CD4+ T cell responses that impair CD8+ T cell function

Negative immunologic regulatory pathways of vaccine vectors suppress antigen-specific CD8+ T cell responses. Rejuvenating viral vectors Adenovirus serotype 5 (Ad5) vaccine vectors elicit mixed responses—they induce protective CD8+ T cells, but these cells may be partially exhausted. Now, Larocca et al. demonstrate that this exhausted phenotype may result from Ad5 vector–induced antigen-specific CD4+ T cells that express interleukin-10 (IL-10) and programmed cell death 1 (PD-1) in both mice and macaques. These IL-10+CD4+ T cells suppress the vaccine-induced CD8+ T cell response, and their inhibitory function may depend in part on IL-27. These data suggest that targeting this inhibitory pathway may enhance protection of viral vector–based vaccines. Adenovirus serotype 5 (Ad5) vaccine vectors elicit robust CD8+ T cell responses, but these responses typically exhibit a partially exhausted phenotype. However, the immunologic mechanism by which Ad5 vectors induce dysfunctional CD8+ T cells has not been elucidated previously. Here, we demonstrate that, after immunization of B6 mice, Ad5 vectors elicit antigen-specific IL-10+CD4+ T cells with a distinct transcriptional profile in a dose-dependent fashion. In rhesus monkeys, we similarly observed up-regulated expression of interleukin-10 (IL-10) and programmed cell death 1 (PD-1) by CD4+ T cells after Ad5 vaccination. These cells markedly suppressed vaccine-elicited CD8+ T cell responses in mice, and IL-10 blockade increased the frequency and functionality of antigen-specific CD8+ T cells, as well as improved protective efficacy against challenge with recombinant Listeria monocytogenes. Moreover, induction of these inhibitory IL-10+CD4+ T cells correlated with IL-27 expression, and IL-27 blockade substantially improved CD4+ T cell functionality. These data highlight a role for IL-27 in the induction of inhibitory IL-10+CD4+ T cells, which suppress CD8+ T cell magnitude and function following Ad5 vector immunization. A deeper understanding of the cytokine networks and transcriptional profiles induced by vaccine vectors should lead to strategies to improve the immunogenicity and protective efficacy of viral vector–based vaccines.

Epitopes for neutralizing antibodies induced by HIV-1 envelope glycoprotein BG505 SOSIP trimers in rabbits and macaques

The native-like, soluble SOSIP.664 trimer based on the BG505 clade A env gene of HIV-1 is immunogenic in various animal species, of which the most studied are rabbits and rhesus macaques. The trimer induces autologous neutralizing antibodies (NAbs) consistently but at a wide range of titers and with incompletely determined specificities. A precise delineation of immunogenic neutralization epitopes on native-like trimers could help strategies to extend the NAb response to heterologous HIV-1 strains. One autologous NAb epitope on the BG505 Env trimer is known to involve residues lining a hole in the glycan shield that is blocked by adding a glycan at either residue 241 or 289. This glycan-hole epitope accounts for the NAb response of most trimer-immunized rabbits but not for that of a substantial subset. Here, we have used a large panel of mutant BG505 Env-pseudotyped viruses to define additional sites. A frequently immunogenic epitope in rabbits is blocked by adding a glycan at residue 465 near the junction of the gp120 V5 loop and β24 strand and is influenced by amino-acid changes in the structurally nearby C3 region. We name this new site the “C3/465 epitope”. Of note is that the C3 region was under selection pressure in the infected infant from whom the BG505 virus was isolated. A third NAb epitope is located in the V1 region of gp120, although it is rarely immunogenic. In macaques, NAb responses induced by BG505 SOSIP trimers are more often directed at the C3/465 epitope than the 241/289-glycan hole.

Development of novel replication-defective lymphocytic choriomeningitis virus vectors expressing SIV antigens

An important focus in vaccine research is the design of vaccine vectors with low seroprevalence and high immunogenicity. Replication-incompetent lymphocytic choriomeningitis virus (rLCMV) vectors do not elicit vector-neutralizing antibody responses, and homologous prime-boost regimens with rLCMV vectors induce boostable and protective T cell responses to model antigens in mice. However, cellular and humoral immune responses following homologous rLCMV vaccine regimens have not been rigorously evaluated in non-human primates (NHPs). To test whether rLCMV vectors constitute an effective vaccine platform in NHPs, we developed rLCMV vectors expressing SIVmac239 Env and Gag antigens and assessed their immunogenicity in mice and cynomolgus macaques. Immunization with rLCMV vaccine vectors expressing SIV Env and Gag was effective at generating SIV-specific T cell and antibody responses in both mice and NHPs. Epitope mapping using SIV Env in C57BL/6 mice demonstrated that rLCMV vectors induced sustained poly-functional responses to both dominant and subdominant epitopes. Our results suggest the potential of rLCMV vectors as vaccine candidates. Future SIV challenge experiments in rhesus macaques will be needed to assess immune protection by these vaccine vectors.