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 attenuated rubella vectors expressing SIV and HIV vaccine antigens replicate and elicit durable immune responses in rhesus macaques.

BACKGROUND: Live attenuated viruses are among our most potent and effective vaccines. For human immunodeficiency virus, however, a live attenuated strain could present substantial safety concerns. We have used the live attenuated rubella vaccine strain RA27/3 as a vector to express SIV and HIV vaccine antigens because its safety and immunogenicity have been demonstrated in millions of children. One dose protects for life against rubella infection. In previous studies, rubella vectors replicated to high titers in cell culture while stably expressing SIV and HIV antigens. Their viability in vivo, however, as well as immunogenicity and antibody persistence, were unknown. RESULTS: This paper reports the first successful trial of rubella vectors in rhesus macaques, in combination with DNA vaccines in a prime and boost strategy. The vectors grew robustly in vivo, and the protein inserts were highly immunogenic. Antibody titers elicited by the SIV Gag vector were greater than or equal to those elicited by natural SIV infection. The antibodies were long lasting, and they were boosted by a second dose of replication-competent rubella vectors given six months later, indicating the induction of memory B cells. CONCLUSIONS: Rubella vectors can serve as a vaccine platform for safe delivery and expression of SIV and HIV antigens. By presenting these antigens in the context of an acute infection, at a high level and for a prolonged duration, these vectors can stimulate a strong and persistent immune response, including maturation of memory B cells. Rhesus macaques will provide an ideal animal model for demonstrating immunogenicity of novel vectors and protection against SIV or SHIV challenge.

Enhanced expression of HIV and SIV vaccine antigens in the structural gene region of live attenuated rubella viral vectors and their incorporation into virions.

Despite the urgent need for an HIV vaccine, its development has been hindered by virus variability, weak immunogenicity of conserved epitopes, and limited durability of the immune response. For other viruses, difficulties with immunogenicity were overcome by developing live attenuated vaccine strains. However, there is no reliable method of attenuation for HIV, and an attenuated strain would risk reversion to wild type. We have developed rubella viral vectors, based on the live attenuated vaccine strain RA27/3, which are capable of expressing important HIV and SIV vaccine antigens. The rubella vaccine strain has demonstrated safety, immunogenicity, and long lasting protection in millions of children. Rubella vectors combine the growth and immunogenicity of live rubella vaccine with the antigenicity of HIV or SIV inserts. This is the first report showing that live attenuated rubella vectors can stably express HIV and SIV vaccine antigens at an insertion site located within the structural gene region. Unlike the Not I site described previously, the new site accommodates a broader range of vaccine antigens without interfering with essential viral functions. In addition, antigens expressed at the structural site were controlled by the strong subgenomic promoter, resulting in higher levels and longer duration of antigen expression. The inserts were expressed as part of the structural polyprotein, processed to free antigen, and incorporated into rubella virions. The rubella vaccine strain readily infects rhesus macaques, and these animals will be the model of choice for testing vector growth in vivo and immunogenicity.

Screening Helicobacter pylori genes induced during infection of mouse stomachs.

AIM: To investigate the effect of in vivo environment on gene expression in Helicobacter pylori (H. pylori) as it relates to its survival in the host. METHODS: In vivo expression technology (IVET) systems are used to identify microbial virulence genes. We modified the IVET-transcriptional fusion vector, pIVET8, which uses antibiotic resistance as the basis for selection of candidate genes in host tissues to develop two unique IVET-promoter-screening vectors, pIVET11 and pIVET12. Our novel IVET systems were developed by the fusion of random Sau3A DNA fragments of H. pylori and a tandem-reporter system of chloramphenicol acetyltransferase and beta-galactosidase. Additionally, each vector contains a kanamycin resistance gene. We used a mouse macrophage cell line, RAW 264.7 and mice, as selective media to identify specific genes that H. pylori expresses in vivo. Gene expression studies were conducted by infecting RAW 264.7 cells with H. pylori. This was followed by real time polymerase chain reaction (PCR) analysis to determine the relative expression levels of in vivo induced genes. RESULTS: In this study, we have identified 31 in vivo induced (ivi) genes in the initial screens. These 31 genes belong to several functional gene families, including several well-known virulence factors that are expressed by the bacterium in infected mouse stomachs. Virulence factors, vacA and cagA, were found in this screen and are known to play important roles in H. pylori infection, colonization and pathogenesis. Their detection validates the efficacy of these screening systems. Some of the identified ivi genes have already been implicated to play an important role in the pathogenesis of H. pylori and other bacterial pathogens such as Escherichia coli and Vibrio cholerae. Transcription profiles of all ivi genes were confirmed by real time PCR analysis of H. pylori RNA isolated from H. pylori infected RAW 264.7 macrophages. We compared the expression profile of H. pylori and RAW 264.7 coculture with that of H. pylori only. Some genes such as cagA, vacA, lpxC, murI, tlpC, trxB, sodB, tnpB, pgi, rbfA and infB showed a 2-20 fold upregulation. Statistically significant upregulation was obtained for all the above mentioned genes (P < 0.05). tlpC, cagA, vacA, sodB, rbfA, infB, tnpB, lpxC and murI were also significantly upregulated (P < 0.01). These data suggest a strong correlation between results obtained in vitro in the macrophage cell line and in the intact animal. CONCLUSION: The positive identification of these genes demonstrates that our IVET systems are powerful tools for studying H. pylori gene expression in the host environment.

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.

Hepatitis B virus surface antigen assembly function persists when entire transmembrane domains 1 and 3 are replaced by a heterologous transmembrane sequence.

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.

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 rubella's 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.

Targeted deletion in the beta20-beta21 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.

Macromolecular crowding stabilizes the molten globule form of apomyoglobin with respect to both cold and heat unfolding.

At pH 2 apomyoglobin is extensively unfolded. Addition of increasing concentration of salts has been shown to convert the protein into molten globule form(s), which can undergo both heat-induced and cold-induced unfolding. Increasing concentrations of an inert polymer, dextran, lead to increased formation of molten globule and stabilizes the protein with respect to both heat-induced and cold-induced denaturation. The transitions were studied by circular dichroism. Two-state analysis of the data shows that the effects of salt and polymer are additive, and that stabilization by the polymer is independent of temperature, as predicted by excluded volume theory.

Protection against scrub typhus by a plasmid vaccine encoding the 56-KD outer membrane protein antigen gene.

The 56-kD outer membrane protein of Orientia tsutsugamushi has previously been shown to be the immunodominant antigen in scrub typhus infections. Its gene was cloned into the DNA vaccine vector pVR1012 as a vaccine candidate (pKarp56). The in vitro expression of this 56-kD antigen by pKarp56 was confirmed in tissue culture by an indirect fluorescence assay and Western blot analysis. The initial antibody responses of mice immunized with varied doses of the pKarp56 were barely detected, but increases were observed after each of three subsequent booster immunizations. Although no protection was observed with a single immunization of pKarp56, after four immunizations, 60% of the mice survived a 1,000 x 50% lethal dose (LD(50)) challenge. These results specifically confirm the importance of the 56-kD protein antigen in protective immunity against O. tsutsugamushi and demonstrate the feasibility of DNA vaccines for the prevention of scrub typhus.