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As the diversity of potential immunogens increases within certain classes of vectors, the possibility has arisen of employing heterologous prime/boost immunizations using diverse members of the same family of vectors. The present study was initiated to explore the use of divergent pox vectors in a prime/boost regimen to elicit high-frequency cellular immune responses to human immunodeficiency virus type 1 envelope and simian immunodeficiency virus gag in rhesus monkeys. We demonstrated that monkeys vaccinated with a recombinant modified vaccinia virus Ankara (rMVA) prime/recombinant fowlpox virus (rFPV) boost regimen and monkeys vaccinated with a recombinant vaccinia virus prime/rFPV boost regimen developed comparable cellular immune responses that were greater in magnitude than those elicited by a homologous prime/boost with rMVA. Nevertheless, comparable magnitude recall cellular immune responses were observed in monkeys vaccinated with heterologous and homologous recombinant poxvirus following challenge with the CXCR4-tropic SHIV-89.6P. Consistent with this finding, comparable levels of containment of viral replication and CD4 + T-lymphocyte preservation were seen in these groups of recombinant poxvirus-vaccinated monkeys. This study supports further exploration of combining recombinant vectors of the same family in prime/boost immunization strategies to optimize vaccine-elicited cellular immune responses.

Rhesus macaques chronically infected with highly pathogenic simian immunodeficiency virus (SIV) SIVmac251 were treated with antiretroviral drugs and vaccinated with combinations of DNA vectors expressing SIV antigens. Vaccination during therapy increased cellular immune responses. After the animals were released from therapy, the virus levels of 12 immunized animals were significantly lower ( P = 0.001) compared to those of 11 animals treated with only antiretroviral drugs. Vaccinated animals showed a persistent increase in immune responses, thus indicating both a virological and an immunological benefit following DNA therapeutic vaccination. Several animals show a long-lasting decrease in viremia, suggesting that therapeutic vaccination may provide an additional benefit to antiretroviral therapy.

Due to the alarming spread of human immunodeficiency virus type 1 (HIV-1) infection worldwide, the development of a prophylactic vaccine is critical. Vaccine strategies for AIDS have included the use of structural and nonstructural HIV subunit proteins or peptides, naked DNA, bacterial and viral live vectors, or combinations of the above. Most of the vaccines developed thus far induce T-cell responses but are unable to induce neutralizing antibodies to the Env of primary HIV-1 isolates. The fact that a decrease in plasma viremia (9, 41) is concomitant with the appearance of virus-specific cytotoxic T lymphocytes (CTLs) and depletion of CD8+ T cells in simian immunodeficiency virus (SIV)-infected macaques or HIV-infected chimpanzees suggests that CD8+ T cells are able to partially control HIV-1/SIV replication (15, 36, 43, 52). Indeed, several “T-cell vaccines” based on DNA and live vectors confer protection from high-level replication of challenge viruses in rhesus macaques (2, 4, 8, 30, 31, 48). The extent of the decrease in viral replication induced by these vaccines is variable in nonhuman primates, and protection from disease appears to be dependent on the virulence of the virus used in the challenge experiments (28). The relative efficacy of poxvirus-based vaccine candidates with various degrees of attenuation has been extensively studied in rhesus macaques after challenge with SIV, SHIV, and HIV-2 isolates (1, 3, 8, 24, 25, 29-31, 48). These vaccine modalities elicit variable levels of cell-mediated immune response and prevent infection after challenge exposure to viruses with low virulence, such as HIV-2 (3, 25), and to other somewhat-attenuated SIV isolates (34, 35). Importantly, these vaccine modalities were also able to significantly reduce virus load after challenge with highly pathogenic SIV isolates (8, 29, 32, 48). An ALVAC-SIV vaccine encoding the gag, pol, and env genes of the SIVmac251 isolate was able to reduce plasma virus load during primary infection and conferred protection from CD4+ loss during both acute and chronic phases of infection after rectal exposure to a highly pathogenic SIVmac251 isolate (50). Among the pox vector-based vaccines, several ALVAC-based HIV-1 vaccines have been tested in phase I and II clinical trials and have been shown to be safe and immunogenic in humans (10, 14, 18, 21, 27). Whether the immunogenicity of these vaccine candidates will be sufficient to protect humans from HIV-1 remains unknown (5, 11, 44). The ongoing human phase III trial in Thailand will provide key information in this regard. Here, we designed a study to assess whether systemic immunization with recombinant canarypox expressing either HIV-1 gp120 or gp160 followed by a boost with either purified gp120 or gp140 proteins would confer protection after mucosal challenge with the pathogenic SHIVKU2. We also investigated whether the addition of the Tat protein of HIV-1 as an immunogen could provide better protection, since a few studies have demonstrated that immunization of macaques with Tat protein either alone or as a Nef-Tat fusion protein in a multicomponent subunit vaccine confers an advantage in a SHIV89.6P model (12, 13, 56) but not in other studies (53). Here, we found that immunization of macaques with this type of ALVAC-based vaccine formulation elicited both antibody and cellular responses and significantly decreased plasma viremia and CD4+ T-cell loss after rectal exposure to the SHIVKU2 isolate. Tat protein immunization had no additive effect on the reduction of virus load in vaccinated rhesus macaques, as also observed by others (42, 46, 53).

Despite extensive efforts made to combat human immunodeficiency virus (HIV) infection and AIDS since the discovery of the virus, the number of people infected with HIV and developing the disease worldwide is still increasing rapidly. The need for a vaccine against HIV is now one of the world's greatest public health problems; however, development of a safe and effective HIV vaccine has proved difficult due to several unique challenges presented by the virus. These include difficulty in eliciting broadly reactive neutralizing antibodies, the high variability of the virus, and integration of HIV proviral DNA into the host genome, resulting in latent infection and making achievement of sterilizing immunity nearly impossible (44). Considering recent reports associating either humoral or cellular immune responses with protection against HIV infection or disease progression, it is difficult to define requirements for protective immunity against HIV (1, 4, 9, 23, 24, 32, 38, 40). Accumulating evidence indicates that an ideal HIV vaccine should induce broad humoral, cellular, and mucosal immunity against multiple viral antigens in order to combat infectious viral particles and HIV-infected cells at any point during infection (19, 25, 33, 50). To achieve this goal, many strategies are being investigated, including recombinant viral proteins and peptides, naked DNA, live viral and bacterial vectors, and prime-boost combinations (19). Adenovirus (Ad) is one of the live viral vectors being developed for use as an HIV vaccine. Ad infects a broad spectrum of human cells, including immature dendritic cells, leading to efficient antigen presentation and causing their maturation without polarizing the T-helper response (22, 53, 54). Because AIDS is mainly a sexually transmitted disease, vaccine-elicited mucosal immunity against HIV is critical. Ad vectors are therefore highly attractive, because they target epithelial cells at mucosal surfaces and can be administered orally and intranasally. Both replication-competent and replication-defective Ad recombinants have been investigated as potential AIDS vaccines. Replication-defective Ad vectors, long used in gene therapy applications, have been adapted for use as HIV vaccines (5, 46, 51). Recent studies with an E1- and E3-deleted Ad5-SIVgag recombinant to immunize rhesus macaques elicited high-frequency SIV p11C-tetramer-positive cells. Following challenge with pathogenic SHIV89.6P the monkeys exhibited significantly reduced viral burdens and were protected against SHIV-induced disease (46). We have taken a different approach, using replication-competent Ad recombinants with deletions only in the E3 region. Because of the inability of human Ad to replicate in most mammalian species, our studies initially were carried out with chimpanzees, which are permissive for Ad replication. Replication-competent, E3 region-deleted Ad-HIVenv and -HIVgag/pro recombinants were investigated and shown to elicit cellular immune responses, antibody responses in mucosal secretions, high-titer serum antibodies able to neutralize both T-cell-line-adapted and primary HIV isolates, and significant protective efficacy (20, 21, 30, 31, 43, 55). Chimpanzees immunized with an Ad-HIVenv priming/gp120 boosting regimen were protected against both low- and high-dose HIV challenges, including challenge with a heterologous primary HIV isolate. The protection elicited was shown to be long lasting. To further develop this approach in a macaque model, we took advantage of an Ad5 host range mutant (Ad5hr) (41) and carried out experiments by using an Ad5hr-SIVsmH4env/rev recombinant shown to replicate in monkey cells in vitro (8). Again by using a recombinant priming/gp120-boosting regimen, we demonstrated that the Ad5hr recombinant also replicated in vivo and elicited SIV-specific cellular immunity and humoral immune responses in serum and secretory fluids (6, 7). This solely envelope-based vaccine achieved a reduction in acute-phase viral burden following intravaginal challenge with pathogenic SIVmac251; however, the viral load began increasing by 8 weeks postchallenge. Accumulating data have shown the potential benefit of incorporating additional viral antigens that elicit strong cellular immune responses into vaccine regimens. Therefore, we added an SIV gag component to the vaccination regimen. We have reported that coadministration of the Ad5hr-SIVsmH4env/rev plus Ad5hr-SIVgag recombinant elicits cellular immune responses to each of the encoded SIV gene products, Env, Gag, and Rev, and that the responses are boosted by a second administration of the same recombinants (35, 52). These recombinant immunizations also prime strong neutralizing antibodies and both IgG and IgA SIV-specific antibodies in secretory fluids (J. Pinczewski, J. Zhao, L. J. Patterson, N. Malkevitch, G. Alvord, and M. Robert-Guroff, unpublished results). Here we have investigated the ability of the dual immunization regimen to protect against an intrarectal challenge with pathogenic SIVmac251 and have explored whether particular immune responses are correlated with the protective outcome. We report improved protective efficacy against a mucosal SIVmac251 challenge, with viremia reduced at both the acute phase and set point of infection. Several immune parameters contributed to the protective outcome.

Vaccine-induced protection of chimpanzees against laboratory-adapted and syncytium-inducing, multiply passaged primary human immunodeficiency virus type 1 (HIV-1) isolates, but not against non-syncytium-inducing, minimally passaged ones, has been demonstrated. Following challenge with such an isolate, HIV-1 5016 , we obtained complete protection in one of three chimpanzees previously protected against low- and high-dose HIV-1 SF2 exposures after immunization with an adenovirus-HIV-1 MN gp160 priming–HIV-1 SF2 gp120 boosting regimen. At challenge, the protected chimpanzee exhibited broad humoral immunity, including neutralizing antibody activity. These results demonstrate the potential of this combination vaccine strategy and suggest that vaccine protection against an HIV isolate relevant to infection of people is feasible.

Primary RNA transcripts from the human immunodeficiency virus type 1 (HIV-1) are processed into mature mRNA by a complex series of splicing events. Viral structural proteins and reverse transcriptase are translated from unspliced or singly spliced transcripts. Proteins which control virus replication, including tat, rev, and nef, are translated from transcripts which are the product of multiple splicing. We have analyzed the composition and relative abundance of the latter transcripts in long-term infected cell lines and in acutely infected peripheral blood cells by amplification with the polymerase chain reaction (PCR) followed by Southern blot, molecular cloning, and DNA sequence analyses. In H9 cells chronically infected with the HIV-1 strain HTLV-IIIB, the predominant of the three kinds of transcripts is those coding for nef. Transcripts with coding potential for rev constituted an intermediate fraction of those analyzed, while those for tat accounted for only a small minority. A similar pattern was observed with Southern blots of PCR-amplified transcripts from peripheral blood lymphocytes acutely infected with HTLV-IIIB. The same general pattern was also observed with PCR-amplified transcripts from peripheral blood monocyte-macrophages infected with an HIV-1 strain (BA-L) able to grow to high titers in macrophages. In these cells, however, the apparent major form of nef transcript contained only the first and third exons of the multiply spliced transcripts and appeared to be generated by either a single or a triple splicing mechanism. As with lymphocytes, tat-specific mRNAs were by far the least abundant. It thus appears that different cell types infected with different strains of HIV-1 maintain a similar balance of expression in which transcripts for nef vastly predominate over those for tat and that those for rev are intermediate in abundance.