Jennifer Schwartz

Balance of cellular and humoral immunity determines the level of protection by HIV vaccines in rhesus macaque models of HIV infection

Significance Our candidate HIV vaccine, a single-chain gp120-CD4 chimera, elicits protection against acquisition of simian-human immunodeficiency virus (SHIV)/simian immunodeficiency virus (SIV) in rhesus macaques. Antibody-dependent cellular cytotoxicity was an inverse correlate of infection risk. However, it is attenuated when antigen-specific T-cell responses exceed a threshold, presumably due to the generation of CD4+ CCR5+ T cells, the preferred cellular targets of SHIV/SIV. Multiple studies strongly suggest that HIV/SIV-specific T-cell responses are a double-edged sword. On one hand, they are required for T-cell help in the protective antibody response. On the other hand, they appear to mitigate protection by creating new targets for viral replication. Determining the balance between protective antibody responses and attenuating T-cell responses is a key challenge confronting HIV vaccine development. A guiding principle for HIV vaccine design has been that cellular and humoral immunity work together to provide the strongest degree of efficacy. However, three efficacy trials of Ad5-vectored HIV vaccines showed no protection. Transmission was increased in two of the trials, suggesting that this vaccine strategy elicited CD4+ T-cell responses that provide more targets for infection, attenuating protection or increasing transmission. The degree to which this problem extends to other HIV vaccine candidates is not known. Here, we show that a gp120-CD4 chimeric subunit protein vaccine (full-length single chain) elicits heterologous protection against simian-human immunodeficiency virus (SHIV) or simian immunodeficiency virus (SIV) acquisition in three independent rhesus macaque repeated low-dose rectal challenge studies with SHIV162P3 or SIVmac251. Protection against acquisition was observed with multiple formulations and challenges. In each study, protection correlated with antibody-dependent cellular cytotoxicity specific for CD4-induced epitopes, provided that the concurrent antivaccine T-cell responses were minimal. Protection was lost in instances when T-cell responses were high or when the requisite antibody titers had declined. Our studies suggest that balance between a protective antibody response and antigen-specific T-cell activation is the critical element to vaccine-mediated protection against HIV. Achieving and sustaining such a balance, while enhancing antibody durability, is the major challenge for HIV vaccine development, regardless of the immunogen or vaccine formulation.

Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein.

Bacterial transport of many sugars, coupled to their phosphorylation, is carried out by the phosphoenolpyruvate (PEP):sugar phosphotransferase system and involves five phosphoryl group transfer reactions. Sugar translocation initiates with the Mg2+-dependent phosphorylation of enzyme I (EI) by PEP. Crystals of Escherichia coli EI were obtained by mixing the protein with Mg2+ and PEP, followed by oxalate, an EI inhibitor. The crystal structure reveals a dimeric protein where each subunit comprises three domains: a domain that binds the partner PEP:sugar phosphotransferase system protein, HPr; a domain that carries the phosphorylated histidine residue, His-189; and a PEP-binding domain. The PEP-binding site is occupied by Mg2+ and oxalate, and the phosphorylated His-189 is in-line for phosphotransfer to/from the ligand. Thus, the structure represents an enzyme intermediate just after phosphotransfer from PEP and before a conformational transition that brings His-189∼P in proximity to the phosphoryl group acceptor, His-15 of HPr. A model of this conformational transition is proposed whereby swiveling around an α-helical linker disengages the His domain from the PEP-binding domain. Assuming that HPr binds to the HPr-binding domain as observed by NMR spectroscopy of an EI fragment, a rotation around two linker segments orients the His domain relative to the HPr-binding domain so that His-189∼P and His-15 are appropriately stationed for an in-line phosphotransfer reaction.

Boosting of ALVAC-SIV Vaccine-Primed Macaques with the CD4-SIVgp120 Fusion Protein Elicits Antibodies to V2 Associated with a Decreased Risk of SIVmac251 Acquisition

The recombinant ALVAC vaccine coupled with the monomeric gp120/alum protein have decreased the risk of HIV and SIV acquisition. Ab responses to the V1/V2 regions have correlated with a decreased risk of virus acquisition in both humans and macaques. We hypothesized that the breadth and functional profile of Abs induced by an ALVAC/envelope protein regimen could be improved by substituting the monomeric gp120 boost, with the full-length single-chain (FLSC) protein. FLSC is a CD4-gp120 fusion immunogen that exposes cryptic gp120 epitopes to the immune system. We compared the immunogenicity and relative efficiency of an ALVAC-SIV vaccine boosted either with bivalent FLSC proteins or with monomeric gp120 in alum. FLSC was superior to monomeric gp120 in directing Abs to the C3 α2 helix, the V5 loop, and the V3 region that contains the putative CCR5 binding site. In addition, FLSC boosting elicited significantly higher binding Abs to V2 and increased both the Ab-dependent cellular cytotoxicity activity and the breadth of neutralizing Abs. However, the FLSC vaccine regimen demonstrated only a trend in vaccine efficacy, whereas the monomeric gp120 regimen significantly decreased the risk of SIVmac251 acquisition. In both vaccine regimens, anti-V2 Abs correlated with a decreased risk of virus acquisition but differed with regard to systemic or mucosal origin. In the FLSC regimen, serum Abs to V2 correlated, whereas in the monomeric gp120 regimen, V2 Abs in rectal secretions, the site of viral challenge, were associated with efficacy.

The catalytic A1 domains of cholera toxin and heat-labile enterotoxin are potent DNA adjuvants that evoke mixed Th1/Th17 cellular immune responses

DNA encoded adjuvants are well known for increasing the magnitude of cellular and/or humoral immune responses directed against vaccine antigens. DNA adjuvants can also tune immune responses directed against vaccine antigens to better protect against infection of the target organism. Two potent DNA adjuvants that have unique abilities to tune immune responses are the catalytic A1 domains of Cholera Toxin (CTA1) and Heat-Labile Enterotoxin (LTA1). Here, we have characterized the adjuvant activities of CTA1 and LTA1 using HIV and SIV genes as model antigens. Both of these adjuvants enhanced the magnitude of antigen-specific cellular immune responses on par with those induced by the well-characterized cytokine adjuvants IL-12 and GM-CSF. CTA1 and LTA1 preferentially enhanced cellular responses to the intracellular antigen SIVmac239-gag over those for the secreted HIVBaL-gp120 antigen. IL-12, GM-CSF and electroporation did the opposite suggesting differences in the mechanisms of actions of these diverse adjuvants. Combinations of CTA1 or LTA1 with IL-12 or GM-CSF generated additive and better balanced cellular responses to both of these antigens. Consistent with observations made with the holotoxin and the CTA1-DD adjuvant, CTA1 and LTA1 evoked mixed Th1/Th17 cellular immune responses. Together, these results show that CTA1 and LTA1 are potent DNA vaccine adjuvants that favor the intracellular antigen gag over the secreted antigen gp120 and evoke mixed Th1/Th17 responses against both of these antigens. The results also indicate that achieving a balanced immune response to multiple intracellular and extracellular antigens delivered via DNA vaccination may require combining adjuvants that have different and complementary mechanisms of action.

An HIV gp120-CD4 Immunogen Does Not Elicit Autoimmune Antibody Responses in Cynomolgus Macaques

ABSTRACT A promising concept for human immunodeficiency virus (HIV) vaccines focuses immunity on the highly conserved transition state structures and epitopes that appear when the HIV glycoprotein gp120 binds to its receptor, CD4. We are developing chimeric antigens (full-length single chain, or FLSC) in which gp120 and CD4 sequences are flexibly linked to allow stable intrachain complex formation between the two moieties (A. DeVico et al., Proc Natl Acad Sci U S A 104:17477–17482, 2007, doi:10.1073/pnas.0707399104; T. R. Fouts et al., J Virol 74:11427–11436, 2000, doi:10.1128/JVI.74.24.11427-11436.2000). Proof of concept studies with nonhuman primates show that FLSC elicited heterologous protection against simian-human immunodeficiency virus (SHIV)/simian immunodeficiency virus (SIV) (T. R. Fouts et al., Proc Natl Acad Sci U S A 112:E992–E999, 2016, doi:10.1073/pnas.1423669112), which correlated with antibodies against transition state gp120 epitopes. Nevertheless, advancement of any vaccine that comprises gp120-CD4 complexes must consider whether the CD4 component breaks tolerance and becomes immunogenic in the autologous host. To address this, we performed an immunotoxicology study with cynomolgus macaques vaccinated with either FLSC or a rhesus variant of FLSC containing macaque CD4 sequences (rhFLSC). Enzyme-linked immunosorbent assay (ELISA) binding titers, primary CD3+ T cell staining, and temporal trends in T cell subset frequencies served to assess whether anti-CD4 autoantibody responses were elicited by vaccination. We find that immunization with multiple high doses of rhFLSC did not elicit detectable antibody titers despite robust responses to rhFLSC. In accordance with these findings, immunized animals had no changes in circulating CD4+ T cell counts or evidence of autoantibody reactivity with cell surface CD4 on primary naive macaque T cells. Collectively, these studies show that antigens using CD4 sequences to stabilize transition state gp120 structures are unlikely to elicit autoimmune antibody responses, supporting the advancement of gp120-CD4 complex-based antigens, such as FLSC, into clinical testing.