Comparison of human and rhesus macaque T-cell responses elicited by boosting with NYVAC encoding human immunodeficiency virus type 1 clade C immunogens.

Rhesus macaques (Macaca mulatta) have played a valuable role in the development of human immunodeficiency virus (HIV) vaccine candidates prior to human clinical trials. However, changes and/or improvements in immunogen quality in the good manufacturing practice (GMP) process or changes in adjuvants, schedule, route, dose, or readouts have compromised the direct comparison of T-cell responses between species. Here we report a comparative study in which T-cell responses from humans and macaques to HIV type 1 antigens (Gag, Pol, Nef, and Env) were induced by the same vaccine batches prepared under GMP and administered according to the same schedules in the absence and presence of priming. Priming with DNA (humans and macaques) or alphavirus (macaques) and boosting with NYVAC induced robust and broad antigen-specific responses, with highly similar Env-specific gamma interferon (IFN-gamma) enzyme-linked immunospot assay responses in rhesus monkeys and human volunteers. Persistent cytokine responses of antigen-specific CD4(+) and CD8(+) T cells of the central memory as well as the effector memory phenotype, capable of simultaneously eliciting multiple cytokines (IFN-gamma, interleukin 2, and tumor necrosis factor alpha), were induced. Responses were highly similar in humans and primates, confirming earlier data indicating that priming is essential for inducing robust NYVAC-boosted IFN-gamma T-cell responses. While significant similarities were observed in Env-specific responses in both species, differences were also observed with respect to responses to other HIV antigens. Future studies with other vaccines using identical lots, immunization schedules, and readouts will establish a broader data set of species similarities and differences with which increased confidence in predicting human responses may be achieved.

Better protective effects in rhesus macaques by combining systemic and mucosal application of a dual component vector vaccine after rectal SHIV89.6P challenge compared to systemic vaccination alone.

In this study we investigated the efficacy of a multigenic DNA prime/modified vaccinia Ankara (MVA)boost vaccine approach, followed by mucosal challenge with highly pathogenic simian-human immunodeficiency virus (SHIV) 89.6P, using different routes for vaccine delivery. After three times of DNA priming (SIVmac239, GagPol, and SHIV 89.6P Env) one vaccine group of monkeys was immunized with MVA systemically via intramuscular (IM) and intradermal (ID) application, and in another vaccine group the MVA booster immunization comprised the IM, ID, and atraumatic oral route. Although all vaccinees became infected after intra-rectal challenge with SHIV 89.6P, substantial protection as indicated by lower peak and set point viral loads and unambiguous preservation of CD4 T cells could be achieved. As we could only transiently detect low levels of neutralizing antibodies in some vaccinees, these antibodies did not seem to add to the protection in the vaccinees. Our results indicate that both preventive multigenic DNA prime/MVA booster immunization strategies promote the control of virus replication and protect from disease progression. We also demonstrated that combining mucosal and systemic vaccination mediated better protective effects compared to systemic vaccination alone.

HIV-1 gag expression is quantitatively dependent on the ratio of native and optimized codons.

There is a significant variation of codon usage bias among different species and even among genes within the same organisms. Codon optimization, this is, gene redesigning with the use of codons preferred for the specific expression system, results in improved expression of heterologous genes in bacteria, plants, yeast, mammalian cells, and transgenic animals. The mechanisms preventing expression of genes with rare or low-usage codons at adequate levels are not completely elucidated. Human immunodeficiency virus (HIV) represents an interesting model for studying how differences in codon usage affect gene expression in heterologous systems. Construction of synthetic genes with optimized codons demonstrated that the codon-usage effects might be a major impediment to the efficient expression of HIV gag/pol and env gene products in mammalian cells. According to another hypothesis, the poor expression of HIV structural proteins even without HIV context is attributed to the so-called cis-acting inhibitory elements (INS), which are located within the protein-coding region. They consist of AU-rich sequences and may be inactivated through the introduction of multiple mutations over the large regions of gag gene. In our work, we evaluated expression of hybrid HIV-1 gag mRNAs where wild-type (A-rich) gag sequences were combined with artificial sequences. In such "humanized" gag fragments with adapted codon usage, AT-content was significantly reduced in favor of G and C nucleotides without any changes in protein sequence. We show that wild-type gag sequences negatively influence expression of gag-reporter, and the addition of fragments with optimized codons to gag mRNA partially rescues its expression. The results demonstrate that the expression of HIV-1 gag is determined by the ratio of optimized and rare codons within mRNA. Our data also indicates that some wtgag fragments counteract the influence of the other wtgag sequences, which cause the inhibition of gag expression. The presented data do not contradict the concept of INS; yet, it makes the definition of INS more complex. This supports the idea of a broader role of the selected codon usage in influencing the expression of HIV proteins in mammalian cells.

Preclinical evaluation of the immunogenicity of C-type HIV-1-based DNA and NYVAC vaccines in the Balb/C mouse model.

As part of a European initiative (EuroVacc), we report the design, construction, and immunogenicity of two HIV-1 vaccine candidates based on a clade C virus strain (CN54) representing the current major epidemic in Asia and parts of Africa. Open reading frames encoding an artificial 160-kDa GagPolNef (GPN) polyprotein and the external glycoprotein gp120 were fully RNA and codon optimized. A DNA vaccine (DNA-GPN and DNA-gp120, referred to as DNA-C), and a replication-deficient vaccinia virus encoding both reading frames (NYVAC-C), were assessed regarding immunogenicity in Balb/C mice. The intramuscular administration of both plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial T-cell responses against both antigens as well as Env-specific antibodies. Whereas low doses of NYVAC-C failed to induce specific CTL or antibodies, high doses generated cellular as well as humoral immune responses, but these did not reach the levels seen following DNA vaccination. The most potent immune responses were detectable using prime:boost protocols, regardless of whether DNA-C or NYVAC-C was used as the priming or boosting agent. These preclinical findings revealed the immunogenic response triggered by DNA-C and its enhancement by combining it with NYVAC-C, thus complementing the macaque preclinical and human phase I clinical studies of EuroVacc.

Capsid stability and replication of human immunodeficiency virus type 1 are influenced critically by charge and size of Gag residue 183.

Structural data support a model where - following proteolytic cleavage--the amino-terminal domain of human immunodeficiency virus type 1 (HIV-1) capsid protein refolds into a beta-hairpin/helix tertiary structure that is stabilized by a buried salt bridge forming between the positively charged primary imino group of a proline residue and the negatively charged carboxyl group of a conserved aspartate. In order to evaluate the contribution of either side-chain length or charge to the formation of infectious virus capsids, aspartate 183 was substituted for glutamate or asparagine in the viral context. It was found that both modifications abolished infectivity of the corresponding viruses in permissive T lymphocytes, although none of particle assembly and release, RNA encapsidation, incorporation of Env glycoproteins and packaging of cyclophilin A were impaired. However, whereas biophysical analyses of mutant virions yielded wild-type-like particle sizes and densities, electron microscopy revealed aberrant core morphologies that could be attributed to either increased (D183N) or reduced (D183E) capsid stability. Although the two amino acid substitutions had opposing effects upon core stability, both mutants were shown to exhibit a severe block in early reverse transcription, underscoring the importance of correct salt-bridge formation for early steps of virus replication.

Generation and immunogenicity of novel HIV/AIDS vaccine candidates targeting HIV-1 Env/Gag-Pol-Nef antigens of clade C.

Recombinants based on the attenuated vaccinia virus strains MVA and NYVAC are considered candidate vectors against different human diseases. In this study we have generated and characterized in BALB/c and in transgenic HHD mice the immunogenicity of two attenuated poxvirus vectors expressing in a single locus (TK) the codon optimized HIV-1 genes encoding gp120 and Gag-Pol-Nef (GPN) polyprotein of clade C (referred as MVA-C and NYVAC-C). In HHD mice primed with either MVA-C or NYVAC-C, or primed with DNA-C and boosted with the poxvirus vectors, the splenic T cell responses against clade C peptides spanning gp120/GPN was broad and mainly directed against Gag-1, Env-1 and Env-2 peptide pools. In BALB/c mice immunized with the homologous or the heterologous combination of poxvirus vectors or with Semliki forest virus (SFV) vectors expressing gp120/GPN, the immune response was also broad but the most immunogenic peptides were Env-1, GPN-1 and GPN-2. Differences in the magnitude of the cellular immune responses were observed between the poxvirus vectors depending on the protocol used. The specific cellular immune response triggered by the poxvirus vectors was Th1 type. The cellular response against the vectors was higher for NYVAC than for MVA in both HHD and BALB/c mice, but differences in viral antigen recognition between the vectors was observed in sera from the poxvirus-immunized animals. These results demonstrate the immunogenic potential of MVA-C and NYVAC-C as novel vaccine candidates against clade C of HIV-1.

Qualitative T-helper responses to multiple viral antigens correlate with vaccine-induced immunity to simian/human immunodeficiency virus infection.

Evidence is accumulating that CD4(+) T-helper (Th) responses play a critical role in facilitating effector responses which are capable of controlling and even preventing human immunodeficiency virus (HIV) infection. The present work was undertaken to determine whether immunization with multiple antigens influenced individual Th responses and increased protection relative to a single antigen. Rhesus macaques were primed with DNA and boosted (immune-stimulating complex-formulated protein) with a combination of regulatory and structural antigens (Tat-Env-Gag) or with Tat alone. Immunization with combined antigens reduced the magnitude of the responses to Tat compared to the single-antigen immunization. Interestingly, the Th immune responses to the individual antigens were noticeably different. To determine whether the qualitative differences in vaccine-induced Th responses correlated with vaccine efficacy, animals were challenged intravenously with simian/human immunodeficiency virus (strain SHIV(89.6p)) 2 months following the final immunization. Animals that developed combined Th1- and Th2-like responses to Gag and Th2 dominant Env-specific responses were protected from disease progression. Interestingly, one animal that was completely protected from infection had the strongest IFN-gamma and interleukin-2 (IL-2) responses prior to challenge, in addition to very strong IL-4 responses to Gag and Env. In contrast, animals with only a marked vaccine-induced Tat-specific Th2 response (no IFN-gamma) were not protected from infection or disease. These data support the rationale that effective HIV vaccine-induced immunity requires a combination of potent Th1- and Th2-like responses best directed to multiple antigens.