Evaluation of cellular immune responses in subjects chronically infected with HIV type 1.

The importance of host cellular immune responses, particularly CD8(+) cytotoxic T-lymphocyte (CTL) responses, in control of human immunodeficiency virus type 1 (HIV-1) infection has been demonstrated in many clinical studies. These studies, along with vaccination challenge studies in rhesus macaques, indicate the importance of cellular immune responses against HIV-1. Toward this end, we evaluated anti-HIV-1 cellular immune responses in a cohort of 54 subjects who were chronically infected with HIV-1. By validation of IFN-gamma ELISpot assay, we established a dual cut-off criterion for scoring a positive response. The magnitude and frequency of cellular immune responses were measured against HIV-1 antigens (Gag, Pol, Nef, Rev, and Tat), using synthetic peptides as antigens in ELISpot assay. Here we showed that HIV-1 Gag, Pol, and Nef were frequent targets of T cell responses in these subjects, whereas Tat and Rev were less frequently recognized. We further evaluated the possible association between host cellular immune responses and corresponding plasma viral loads in this cohort. By performing ranking correlation analysis, we demonstrated a positive correlation between host viral loads and ELISpot responses of HIV Gag and Pol in untreated subjects. For the subjects under antiviral regimens, however, we did not find any significant association. Our findings suggest that the high levels of ELISpot responses in chronically infected subjects were reflective of their persistent viral infection.

Intracellular staining for HIV-specific IFN-gamma production: statistical analyses establish reproducibility and criteria for distinguishing positive responses.

The cellular immune response plays a pivotal role in controlling the spread of HIV-1 infection by lysing virally infected cells and producing potent antiviral cytokines, such as interferon-gamma (IFN-gamma). Flow cytometric methods have been established to evaluate the contribution of both CD4 and CD8 subsets of T lymphocytes to the immune response to HIV by measuring their production of intracellular IFN-gamma following brief antigenic stimulation. We present a statistical treatment of intracellular cytokine staining (ICS) data that is aimed at establishing the reproducibility and robustness of this assay for use in HIV clinical trials. Comparisons of responses from HIV-seronegative and seropositive individuals were used to establish a 2-fold criterion for distinguishing positive responses with a low probability of false positives (<1%). Additional comparisons established that the reproducibility of the assay is between 1.4 and 2.0-fold depending on the magnitude of the response. Little variability was demonstrated between multiple operators for both the execution and analysis components of these experiments (<10% difference with 95% confidence). We conclude that the statistical criteria established by these analyses allow for the accurate detection and comparison of positive responses. Using these statistical criteria, the ICS assay is sufficiently robust for use in HIV-specific vaccine trials.

Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene.

Cellular immune responses, particularly those associated with CD3(+) CD8(+) cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4(+) and CD8(+) T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.

Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity.

Recent studies of human immunodeficiency virus type 1 (HIV-1) infection in humans and of simian immunodeficiency virus (SIV) in rhesus monkeys have shown that resolution of the acute viral infection and control of the subsequent persistent infection are mediated by the antiviral cellular immune response. We comparatively assessed several vaccine vector delivery systems-three formulations of a plasmid DNA vector, the modified vaccinia Ankara (MVA) virus, and a replication incompetent adenovirus type 5 (Ad5) vector-expressing the SIV gag protein for their ability to elicit such immune responses in monkeys. The vaccines were tested either as a single modality or in combined modality regimens. Here we show that the most effective responses were elicited by a replication-incompetent Ad5 vector, used either alone or as a booster inoculation after priming with a DNA vector. After challenge with a pathogenic HIV-SIV hybrid virus (SHIV), the animals immunized with Ad5 vector exhibited the most pronounced attenuation of the virus infection. The replication-defective adenovirus is a promising vaccine vector for development of an HIV-1 vaccine.

Vaccine-induced immune responses in rodents and nonhuman primates by use of a humanized human immunodeficiency virus type 1 pol gene.

A synthetic gene consisting of the reverse transcriptase (RT) and integrase (IN) domains of human immunodeficiency virus type 1 (HIV-1) pol was constructed using codons most frequently used in humans. The humanized pol gave dramatically improved levels of Rev-independent, in vitro protein production in mammalian cells and elicited much stronger cellular immunity in rodents than did virus-derived gene. Specifically, BALB/c mice were immunized with plasmids and/or recombinant vaccinia virus constructs expressing the synthetic gene. High frequencies of Pol-specific T lymphocytes were detected in these animals by the gamma interferon enzyme-linked immunospot assay against pools of short overlapping peptides. Characterization of the stimulatory peptides from these pools indicates that the optimized gene constructs are able to effectively activate both CD4+ and CD8+ T cells. Immunization of rhesus macaques with DNA vaccines expressing the humanized pol coupled to a human tissue plasminogen activator leader sequence led to pronounced in vitro cytotoxic T-lymphocyte killing activities and enhanced levels of circulating Pol-specific T cells, comparable to those observed in HIV-1-infected human subjects. Thus, optimizing the immunogenic properties of HIV-1 Pol at the level of the gene sequence validates it as an antigen and provides an important step toward the construction of a potent pol-based HIV-1 vaccine component.