Ira Berkower

Antibodies to the A27 Protein of Vaccinia Virus Neutralize and Protect against Infection but Represent a Minor Component of Dryvax Vaccine-Induced Immunity

The smallpox vaccine Dryvax, which consists of replication-competent vaccinia virus, elicits antibodies that play a major role in protection. Several vaccinia proteins generate neutralizing antibodies, but their importance for protection is unknown. We investigated the potency of antibodies to the A27 protein of the mature virion in neutralization and protection experiments and the contributions of A27 antibodies to Dryvax-induced immunity. Using a recombinant A27 protein (rA27), we confirmed that A27 contains neutralizing determinants and that vaccinia immune globulin (VIG) derived from Dryvax recipients contains reactivity to A27. However, VIG neutralization was not significantly reduced when A27 antibodies were removed, and antibodies elicited by an rA27 enhanced the protection conferred by VIG in passive transfer experiments. These findings demonstrate that A27 antibodies do not represent the major fraction of neutralizing activity in VIG and suggest that immunity may be augmented by vaccines and immune globulins that include strong antibody responses to A27.

Targeted deletion in the β20-β21 loop of HIV envelope glycoprotein gp120 exposes the CD4 binding site for antibody binding

Different isolates of HIV-1 are known to vary in antibody binding and sensitivity to neutralization. In response to selective pressure, the virus may conceal important neutralizing determinants, such as the CD4 binding site on gp120, through steric hindrance or conformational masking. The 3D structure of gp120 shows five loop structures that surround the CD4 binding site (CD4BS) and may restrict antibody access to the site. We have generated gp120 mutants lacking each of these loops and characterized them with a panel of monoclonal antibodies, including b12 and F105. A targeted deletion in the beta20-beta21 loop resulted in gp120 with enhanced binding of both monoclonals. Enhancement of b12 binding suggests reduced steric hindrance, since the antibody is relatively insensitive to conformation. Enhanced binding of F105, which depends strongly on the protein conformation, suggests that the mutation may allow gp120 to move more freely into the liganded form. The same viral strategies that limit antibody binding may also inhibit antibody induction. Modified forms of gp120, in which the CD4 binding site is more exposed and accessible to antibodies, could provide novel immunogens for eliciting antibodies to this broadly shared neutralizing determinant.

Live attenuated rubella viral vectors stably express HIV and SIV vaccine antigens while reaching high titers.

Live attenuated viruses make potent and effective vaccines. Despite the urgent need for an HIV vaccine, this approach has not been feasible, since it has not been possible to attenuate the virus reliably and guarantee vaccine safety. Instead, live viral vectors have been proposed that could present HIV vaccine antigens in the most immunogenic way, in the context of an active infection. We have adapted the rubella vaccine strain RA27/3 as a vector to express HIV and SIV antigens, and tested the effect of insert size and composition on vector stability and viral titer. We have identified an acceptor site in the rubella nonstructural gene region, where foreign genes can be expressed as a fusion protein with the nonstructural protein P150 without affecting essential viral functions. The inserts were expressed as early genes of rubella, under control of the rubella genomic promoter. At this site, HIV and SIV antigens were expressed stably for at least seven passages, as the rubella vectors reached high titers. Rubella readily infects rhesus macaques, and these animals will provide an ideal model for testing the new vectors for replication in vivo, immunogenicity, and protection against SIV or SHIV challenge.

Stable expression of a foreign protein by a replication-competent rubella viral vector.

Live, attenuated rubella vaccine has been used successfully for many years. By expressing additional viral antigens in rubella, we could expand its range and utility as a live, replicating viral vector. Previously, limitations on insert size and stability restricted rubellas ability to express exogenous antigens and immunize against other viruses. In this study, we have overcome this problem by creating a deletion in non-structural protein P150 that makes room for the insert. The resulting rubella hybrid stably expressed a model protein for over 10 passages, while replicating and expressing rubella proteins normally. The foreign protein, GFP, was as large as many important viral antigens, and the virus grew to sufficiently high titers for vaccine use. Further progress in expressing exogenous viral antigens in rubella may produce live viral vectors capable of immunizing against viruses for which attenuation is not currently feasible.

Hepatitis B Virus Surface Antigen Assembly Function Persists when Entire Transmembrane Domains 1 and 3 Are Replaced by a Heterologous Transmembrane Sequence

ABSTRACT Native hepatitis B surface antigen (HBsAg) spontaneously assembles into 22-nm subviral particles. The particles are lipoprotein micelles, in which HBsAg is believed to span the lipid layer four times. The first two transmembrane domains, TM1 and TM2, are required for particle assembly. We have probed the requirements for particle assembly by replacing the entire first or third TM domain of HBsAg with the transmembrane domain of HIV gp41. We found that either TM domain of HBsAg could be replaced, resulting in HBsAg-gp41 chimeras that formed particles efficiently. HBsAg formed particles even when both TM1 and TM3 were replaced with the gp41 domain. The results indicate remarkable flexibility in HBsAg particle formation and provide a novel way to express heterologous membrane proteins that are anchored to a lipid surface by their own membrane-spanning domain. The membrane-proximal exposed region (MPER) of gp41 is an important target of broadly reactive neutralizing antibodies against HIV-1, and HBsAg-MPER particles may provide a good platform for future vaccine development.

Recombinant rubella vectors elicit SIV Gag-specific T cell responses with cytotoxic potential in rhesus macaques.

Live-attenuated rubella vaccine strain RA27/3 has been demonstrated to be safe and immunogenic in millions of children. The vaccine strain was used to insert SIV gag sequences and the resulting rubella vectors were tested in rhesus macaques alone and together with SIV gag DNA in different vaccine prime-boost combinations. We previously reported that such rubella vectors induce robust and durable SIV-specific humoral immune responses in macaques. Here, we report that recombinant rubella vectors elicit robust de novo SIV-specific cellular immune responses detectable for >10 months even after a single vaccination. The antigen-specific responses induced by the rubella vector include central and effector memory CD4(+) and CD8(+) T cells with cytotoxic potential. Rubella vectors can be administered repeatedly even after vaccination with the rubella vaccine strain RA27/3. Vaccine regimens including rubella vector and SIV gag DNA in different prime-boost combinations resulted in robust long-lasting cellular responses with significant increase of cellular responses upon boost. Rubella vectors provide a potent platform for inducing HIV-specific immunity that can be combined with DNA in a prime-boost regimen to elicit durable cellular immunity.

Expression of complete SIV p27 Gag and HIV gp120 engineered outer domains targeted by broadly neutralizing antibodies in live rubella vectors.

Infection with HIV or SIV often elicits a potent immune response to viral antigens. This includes T cells and antibodies specific for Gag and Env antigens. In contrast, when given as a vaccine, the same antigens have been weak immunogens, unable to elicit antibodies with comparable titer, durability, or neutralizing activity. We have used the live attenuated rubella vaccine strain RA27/3 as a viral vector to express HIV and SIV antigens. By mimicking an HIV infection, these vectors could elicit stronger and more durable immunity to HIV antigens. The vectors are based on the licensed rubella vaccine strain, which has demonstrated safety and potency in millions of children. One or two doses protect for life against rubella infection. The question was whether rubella vectors could similarly enhance the immunogenicity of a foreign vaccine insert. We have previously reported that rubella vectors can express small protein antigens in vitro and in vivo, where they elicit a strong immune response to the vaccine insert. The vectors have now expressed larger vaccine inserts that include epitope-rich fragments of the Gag matrix and capsid proteins (aa 41-211) or the complete p27 capsid protein with p2 (aa 136-381). These vectors have elicited a robust and durable immune response to Gag in rhesus macaques. This size range also encompasses the engineered outer domain (eOD) of HIV envelope gp120 (172 amino acids). The rubella/eOD-GT6 and GT8 vectors stably expressed glycoproteins that bind germline precursors and mature forms of VRC01-class broadly neutralizing antibodies. These vectors potentially could be used as part of a sequential immunization strategy to initiate the production of broadly neutralizing antibodies.

Live rubella vectors can express native HIV envelope glycoproteins targeted by broadly neutralizing antibodies and prime the immune response to an envelope protein boost

Following HIV infection, most people make antibodies to gp120 and gp41, yet only a few make broadly neutralizing antibodies that target key antigenic sites on the envelope glycoproteins. The induction of broadly neutralizing antibodies by immunization remains a major challenge of HIV vaccine research. Difficulties include: variable protein sequence, epitopes that depend on the native conformation, glycosylation that conceals key antigenic determinants, and the assembly of Env trimers that mimic viral spikes. In addition, more potent immunogens may be needed to initiate the response of germline antibody precursors and drive B cell maturation toward antibodies with broad neutralizing activity. We have expressed HIV Env glycoproteins by incorporation into live attenuated rubella viral vectors. The rubella vaccine strain RA27/3 has demonstrated its safety and potency in millions of children. As a vector, it has elicited potent and durable immune responses in macaques to SIV Gag vaccine inserts. We now find that rubella/env vectors can stably express Env core derived glycoproteins ranging in size up to 363 amino acids from HIV clade C strain 426c. The expressed Env glycoproteins bind broadly neutralizing antibodies that target the native CD4 binding site. The vectors grew well in rhesus macaques, and they elicited a vaccine take in all animals, as measured by anti-rubella antibodies. By themselves, the vectors elicited modest antibody titers to the Env insert. But the combination of rubella/env prime followed by a homologous protein boost gave a strong response. Neutralizing antibodies appeared gradually after multiple vaccine doses. The vectors will be useful for testing new vaccine inserts and immunization strategies under optimized conditions of vector growth and protein expression.