Kurt Bieler

Concerted Action of Multiple cis-Acting Sequences Is Required for Rev Dependence of Late Human Immunodeficiency Virus Type 1 Gene Expression

ABSTRACT Based on the human immunodeficiency virus type 1 (HIV-1)gag gene, subgenomic reporter constructs have been established allowing the contributions of differentcis-acting elements to the Rev dependency of late HIV-1 gene products to be determined. Modification of intragenic regulatory elements achieved by adapting the codon usage of the complete gene to highly expressed mammalian genes resulted in constitutive nuclear export allowing high levels of Gag expression independent from the Rev/Rev-responsive element system and irrespective of the absence or presence of the isolated major splice donor. Leptomycin B inhibitor studies revealed that the RNAs derived from the codon-optimizedgag gene lacking AU-rich inhibitory elements are directed to a distinct, CRM1-independent, nuclear export pathway.

Qualitative T-Helper Responses to Multiple Viral Antigens Correlate with Vaccine-Induced Immunity to Simian/Human Immunodeficiency Virus Infection

ABSTRACT 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 SHIV89.6p) 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-γ 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-γ) 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.

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

ABSTRACT 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-γ) 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-γ, 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-γ 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.

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.

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.