Live attenuated rubella vectors expressing Plasmodium falciparum circumsporozoite protein (Pf-CSP) provide a novel malaria vaccine platform in the rhesus macaque.

There is an urgent need for a malaria vaccine that can prevent severe disease in young children and adults. Despite earlier work showing an immunological mechanism for preventing infection and reducing disease severity, there is currently no reliable vaccine that can provide durable protection. In part, this may reflect a limited number of ways that the host can respond to the NANP repeat sequences of circumsporozoite protein (CSP) in the parasite. In addition, it may reflect antigenic escape by the parasite from protective antibodies. To be successful, a vaccine must protect against repeated exposure to infected mosquitoes in endemic areas. We have created a series of live viral vectors based on the rubella vaccine strain that express multiple tandem repeats of NANP, and we demonstrate immunogenicity in a rhesus macaque model. We tested the vectors in a sequential immunization strategy. In the first step, the animals were primed with CSP-DNA vaccine and boosted with rubella/CSP vectors. In the second step, we gave rubella/CSP vectors again, followed by recombinant CSP protein. Following the second step, antibody titers were comparable to adult exposure to malaria in an endemic area. The antibodies were specific for native CSP protein on sporozoites, and they persisted for at least 1½ years in two out of three macaques. Given the safety profile of rubella vaccine in children, these vectors could be most useful in protecting young children, who are at greatest risk of severe malarial disease.

Organization of DNA in a bacterial nucleoid.

BACKGROUND: It is unclear how DNA is packaged in a bacterial cell in the absence of nucleosomes. To investigate the initial level of DNA condensation in bacterial nucleoid we used in vivo DNA digestion coupled with high-throughput sequencing of the digestion-resistant fragments. To this end, we transformed E. coli cells with a plasmid expressing micrococcal nuclease. The nuclease expression was under the control of AraC repressor, which enabled us to perform an inducible digestion of bacterial nucleoid inside a living cell. RESULTS: Analysis of the genomic localization of the digestion-resistant fragments revealed their non-random distribution. The patterns observed in the distribution of the sequenced fragments indicate the presence of short DNA segments protected from the enzyme digestion, possibly because of interaction with DNA-binding proteins. The average length of such digestion-resistant segments is about 50 bp and the characteristic repeat in their distribution is about 90 bp. The gene starts are depleted of the digestion-resistant fragments, suggesting that these genomic regions are more exposed than genomic sequences on average. Sequence analysis of the digestion-resistant segments showed that while the GC-content of such sequences is close to the genome-wide value, they are depleted of A-tracts as compared to the bulk genomic DNA or to the randomized sequence of the same nucleotide composition. CONCLUSIONS: Our results suggest that DNA is packaged in the bacterial nucleoid in a non-random way that facilitates interaction of the DNA binding factors with regulatory regions of the genome.

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.

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.

A gamut of loops: meandering DNA.

Nucleoprotein complexes comprising short DNA loops (150 base pairs or less) are involved in a wide variety of DNA transactions (e.g. transcription regulation, replication and recombination) in both prokaryotes and eukaryotes, and also can be useful in designing nanostructures. In these higher-order nucleoprotein complexes, proteins bound to spatially separated sites on a DNA interact with each other by looping out the relatively stiff intervening DNA. Recent technological developments have enabled determination of DNA trajectories in a few DNA-loop-containing regulatory complexes. Results show that, in a given system, a specific DNA trajectory is preferred over others.

Immunotherapy with DNA vaccine and live attenuated rubella/SIV gag vectors plus early ART can prevent SIVmac251 viral rebound in acutely infected rhesus macaques.

Anti-retroviral therapy (ART) has been highly successful in controlling HIV replication, reducing viral burden, and preventing both progression to AIDS and viral transmission. Yet, ART alone cannot cure the infection. Even after years of successful therapy, ART withdrawal leads inevitably to viral rebound within a few weeks or months. Our hypothesis: effective therapy must control both the replicating virus pool and the reactivatable latent viral reservoir. To do this, we have combined ART and immunotherapy to attack both viral pools simultaneously. The vaccine regimen consisted of DNA vaccine expressing SIV Gag, followed by a boost with live attenuated rubella/gag vectors. The vectors grow well in rhesus macaques, and they are potent immunogens when used in a prime and boost strategy. We infected rhesus macaques by high dose mucosal challenge with virulent SIVmac251 and waited three days to allow viral dissemination and establishment of a reactivatable viral reservoir before starting ART. While on ART, the control group received control DNA and empty rubella vaccine, while the immunotherapy group received DNA/gag prime, followed by boosts with rubella vectors expressing SIV gag over 27 weeks. Both groups had a vaccine "take" to rubella, and the vaccine group developed antibodies and T cells specific for Gag. Five weeks after the last immunization, we stopped ART and monitored virus rebound. All four control animals eventually had a viral rebound, and two were euthanized for AIDS. One control macaque did not rebound until 2 years after ART release. In contrast, there was only one viral rebound in the vaccine group. Three out of four vaccinees had no viral rebound, even after CD8 depletion, and they remain in drug-free viral remission more than 2.5 years later. The strategy of early ART combined with immunotherapy can produce a sustained SIV remission in macaques and may be relevant for immunotherapy of HIV in humans.

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.

Three-stage regulation of the amphibolic gal operon: from repressosome to GalR-free DNA.

The gal operon of Escherichia coli is negatively regulated by the Gal repressosome, a higher order nucleoprotein complex containing a DNA loop that encompasses two gal promoters. In the repressosome structure, Gal repressor (GalR) dimers are bound to the two operator sites, flanking the promoter region, thus generating a DNA loop. The DNA loop is stabilized by binding of the architectural HU protein to the apex of the loop, and negative supercoiling. The gal promoters are also regulated in opposite directions by GalR without DNA looping. The repressosome-mediated as well as looping-independent transcription regulation of the two promoters is lifted in the presence of the inducer D-galactose. We tested the effect of D-galactose on various DNA-protein and protein-protein interactions of different regulatory complexes and on transcription repression in vitro. We found that the inducer breaks up the repressosome with clear intermediates in a concentration-dependent manner. The sequential disassembly generates different stages of regulation of the gal operon. The D-galactose-dependent switch from one stage of regulation to another satisfies the amphibolic requirement of the gal operon.

A-tract clusters may facilitate DNA packaging in bacterial nucleoid.

Molecular mechanisms of bacterial chromosome packaging are still unclear, as bacteria lack nucleosomes or other apparent basic elements of DNA compaction. Among the factors facilitating DNA condensation may be a propensity of the DNA molecule for folding due to its intrinsic curvature. As suggested previously, the sequence correlations in genome reflect such a propensity [Trifonov and Sussman (1980) Proc. Natl Acad. Sci. USA, 77, 3816-3820]. To further elaborate this concept, we analyzed positioning of A-tracts (the sequence motifs introducing the most pronounced DNA curvature) in the Escherichia coli genome. First, we observed that the A-tracts are over-represented and distributed 'quasi-regularly' throughout the genome, including both the coding and intergenic sequences. Second, there is a 10-12 bp periodicity in the A-tract positioning indicating that the A-tracts are phased with respect to the DNA helical repeat. Third, the phased A-tracts are organized in approximately 100 bp long clusters. The latter feature was revealed with the help of a novel approach based on the Fourier series expansion of the A-tract distance autocorrelation function. Since the A-tracts introduce local bends of the DNA duplex and these bends accumulate when properly phased, the observed clusters would facilitate DNA looping. Also, such clusters may serve as binding sites for the nucleoid-associated proteins that have affinities for curved DNA (such as HU, H-NS, Hfq and CbpA). Therefore, we suggest that the approximately 100 bp long clusters of the phased A-tracts constitute the 'structural code' for DNA compaction by providing the long-range intrinsic curvature and increasing stability of the DNA complexes with architectural proteins.

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.

"Antiparallel" DNA loop in gal repressosome visualized by atomic force microscopy.

DNA looping is often involved in positive and negative regulation of gene transcription in both prokaryotes and eukaryotes. The transcription of the gal operon of Escherichia coli from two overlapping promoters P1 and P2 is negatively regulated via Gal repressosome assembly. It involves binding of two dimeric Gal repressor proteins (GalR) to two operators, O(E) and O(I), flanking the two promoters, and formation of 113 bp DNA loop due to tetramerization of the two bound GalR dimers. The process requires negatively supercoiled DNA and the presence of the histone-like protein HU. Previous modeling of the repressosome based on evaluation of DNA elastic energy suggested a mutual antiparallel, rather than parallel, orientation of the two gal operators in an under-twisted DNA loop. To visualize the Gal loop by atomic force microscopy (AFM), plasmid DNA molecules were constructed with increased distance between the two operators. The AFM results demonstrated the formation of an antiparallel DNA loop in the Gal repressosome consistent with our earlier hypothesis. Importantly, the overall shape of the GalR mediated loop proved to be indistinguishable from that in the chimerical loop of the same size containing two lac operators (instead of two gal operators) and formed by LacI. In addition, a possibility of the gal operon repression mediated by GalR in the absence of HU was shown in the new DNA constructs. Implications of these findings for the DNA structural organization in bacterial nucleoid are discussed.

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.

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.

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 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.

DNA trajectory in the Gal repressosome.

The Gal repressosome is a higher-order nucleoprotein complex that represses transcription of the gal operon in Escherichia coli. During the repressosome assembly, a DNA loop is formed by the interaction of two GalR dimers, bound to two spatially separated operators, OE and OI, flanking the gal promoters. Structure-based genetic analysis indicated that GalR homodimers interact directly and form a V-shaped stacked tetramer in repressosome, further stabilized by HU binding to an architecturally critical position on the DNA. In this scheme of GalR tetramerization, the alignment of the operators in the DNA loop could be in either parallel (PL) or antiparallel (AL) mode. As each mode can have two alternative geometries differing in the mutual stacking of the OE- and OI-bound GalR dimers, it is possible to have four different DNA trajectories in the repressosome. Feasibilities of these trajectories were tested by in vitro transcription repression assays, first by isolating GalR mutants with altered operator specificity and then by constructing four different potential loops with mutant GalR heterodimers bound to specifically designed hybrid operators in such a way as to give rise to only one of the four putative trajectories. Results show that OE and OI adopt a mutual antiparallel orientation in an under-twisted DNA loop, consistent with the energetically optimal structural model. In this structure the center of the HU-binding site is located at the apex of the DNA loop. The approach reported here can be used to distinguish between otherwise indistinguishable DNA trajectories in complex nucleoprotein machines.