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The prophylactic efficacy of DNA and replication-incompetent adenovirus serotype 5 (Ad5) vaccine vectors expressing simian immunodeficiency virus (SIV) Gag was examined in rhesus macaques using an SIVmac239 challenge. Cohorts of either Mamu-A*01(+) or Mamu-A*01(−) macaques were immunized with a DNA prime-Ad5 boost regimen; for comparison, a third cohort consisting of Mamu-A*01(+) monkeys was immunized using the Ad5 vector alone for both prime and boost. All animals, along with unvaccinated control cohorts of Mamu-A*01(+) and Mamu-A*01(−) macaques, were challenged intrarectally with SIVmac239. Viral loads were measured in both peripheral and lymphoid compartments. Only the DNA prime-Ad5-boosted Mamu-A*01(+) cohort exhibited a notable reduction in peak plasma viral load (sevenfold) as well as in early set-point viral burdens in both plasma and lymphoid tissues (10-fold) relative to those observed in the control monkeys sharing the same Mamu-A*01 allele. The degree of control in each animal correlated with the levels of Gag-specific immunity before virus challenge. However, virus control was short-lived, and indications of viral escape were evident as early as 6 months postinfection. The implications of these results in vaccine design and clinical testing are discussed.

We compared the human immunodeficiency virus type 1 (HIV-1)-specific cellular immune responses elicited in nonhuman primates by HIV-1 gag -expressing replication-defective adenovirus serotype 5 (Ad5) or poxvirus vectors, used either alone or in combination with each other. The responses arising from a heterologous Ad5 priming-poxvirus boosting regimen were significantly greater than those elicited by homologous regimens with the individual vectors or by a heterologous poxvirus priming-Ad5 boosting regimen. The heterologous Ad5 priming-poxvirus boosting approach may have potential utility in humans as a means of inducing high levels of cellular immunity.

Set-point viral load is positively correlated with the extent of initial viral replication in pathogenic simian-human immunodeficiency virus (SHIV) infection. To elucidate the mechanisms underlying the correlation, we conducted a systematic investigation in rhesus monkeys infected with the highly pathogenic SHIV 89.6P. This model is widely used in the preclinical evaluation of AIDS vaccine candidates and a thorough understanding of the model's biology is important to the proper interpretation of these evaluations. We found that the levels of peak viremia were positively correlated not only with the levels of set-point viremia but, importantly, with the extent of initial overall immune destruction as indicated by the degree of CD4+ T cell depletion and lymph node germinal center (GC) formation. The extent of initial overall immune destruction was inversely correlated with subsequent development and maintenance of virus-specific cellular and humoral immune responses. Thus, these data suggest that the extent of early immune damage determines the development and durability of virus-specific immunity, thereby playing a critical role in establishing the levels of set-point viral replication in SHIV infection. Vaccines that limit both the initial viral replication and the extent of early immune damage will therefore mediate long-term virus replication control and mitigation of long-term immune destruction in this model of immunodeficiency virus infection.

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.

Three-hundred and seventy-six strains of Streptococcus pneumoniae isolated from clinical specimens and nasopharyngeal swabs from children at daycare centers and hospitals in Beijing China, between January 1997 and March 1998, were serotyped. Twenty-seven different serotypes were identified. The most prevalent serotypes in the carriage isolates were 6A, 19F, 23F, and 15 and were found in 66.8% of cases. Serotype data indicate that 51.8% of carrier strains would be included in the 11-valent conjugate vaccine formulation, while inclusion of vaccine-related serotypes, increased the potential vaccine coverage to 79.4%. Serotypes 7, 6B, 23F, 19F, 15, and 3 accounted for 62% of clinical strains, with 70% vaccine-related serotypes. DNA fingerprinting of 47 penicillin resistant and 71 penicillin-susceptible/macrolide-resistant strains by BOX polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE), and penicillin binding protein (PBP)-fingerprinting identified two novel clones: one a serotype 23F multiresistant clone resistant to penicillin, tetracycline, erythromycin, clindamycin, and variably resistant to chloramphenicol and trimethoprim-sulphamethoxazole; and the second a multiresistant penicillin-susceptible, macrolide-resistant serotype 6A clone, highly resistant also to tetracycline, clindamycin, and trimethoprim-sulphamethoxazole. The macrolide resistance determinant in 89% of erythromycin-resistant strains tested (penicillin-susceptible and penicillin-resistant) was the erm gene, both the erm and mef genes were simultaneously found in 6%, and mef alone in 3.4%. The data demonstrates that macrolide resistant strains in China include clonal strains and strains with dual mef and erm resistance determinants.

Safe and efficient methods for in vivo delivery of transgenes of interest must be developed so that the promise of these therapies can be practically used in the clinic. In this work, we describe the use of electrostimulation to enhance the in vivo efficiency of plasmid DNA delivery. The method was optimized to work over a range of moderate frequencies, utilizing low field strengths and simple symmetrical waveforms. After studying several parameters of delivery in mice, we demonstrate how this methodology can be employed to significantly improve both gene expression (over 16-fold) and the immunogenicity of HIV-1 vaccines (over 28-fold) compared to naked DNA in non-human primates. Compared to an efficient viral Ad5 vector system, the gene expression levels of DNA + electrostimulation were surprisingly within a factor of four of the viral delivery system.

Lack of virus specific antibody response is commonly observed in both HIV-1-infected humans and SIV-infected monkeys with rapid diseaseprogression. However, the mechanisms underlying this important observation still remain unclear. In a titration study of a SIVmac239 viral stock,three out of six animals with viral inoculation rapidly progressed to AIDS within 5 months. Unexpectedly, there was no obvious depletion ofCD4 + T cells in both peripheral and lymph node (LN) compartments in these animals. Instead, progressive depletion of proliferating B cells anddisruption of the follicular dendritic cell (FDC) network in germinal centers (GC) was evident in the samples collected at as early as 20 days afterviral challenge. This coincided with undetectable, or weak and transient, virus-specific antibody responses over the course of infection. In situhybridization of SIV RNA in the LN samples revealed a high frequency of SIV productively infected cells and large amounts of accumulated viralRNA in the GCs in these animals. Early severe depletion of GC proliferating B cells and disruption of the FDC network may thus result in aninability to mount a virus-specific antibody response in rapid progressors, which has been shown to contribute to accelerated disease progressionof SIV infection.© 2006 Elsevier Inc. All rights reserved.

Simian-human immunodeficiency virus (SHIV) challenge studies in rhesus macaques were conducted to evaluate the efficacy of adenovirus-based vaccines in the context of different major histocompatibility complex class I genetic backgrounds and different vaccine compositions. Mamu-A * 01 allele-negative rhesus monkeys were immunized with one of the following vaccine constructs: (i) replication-defective recombinant adenovirus type 5 (Ad5) expressing human immunodeficiency virus type 1 (HIV-1) Tat (Ad5/HIVTat); (ii) Ad5 vector expressing simian immunodeficiency virus (SIV) Gag (Ad5/SIVGag); (iii) Ad5 vector expressing the truncated HIV-1 jrfl Env, gp140 (Ad5/gp140_jrfl); (iv) Ad5 vector expressing the SHIV-89.6P gp140 (Ad5/gp140_89.6P); or (v) the combination of Ad5/SIVGag and Ad5/gp140_jrfl. Following intravenous challenge with SHIV-89.6P, only those cohorts that received vaccines expressing Gag or Env exhibited an attenuation of the acute viremia and associated CD4-cell lymphopenia. While no prechallenge neutralizing antibody titers were detectable in either Ad5/gp140-vaccinated group, an accelerated neutralizing antibody response was observed in the Ad5/gp140_89.6P-vaccinated group upon viral challenge. The set-point viral loads in the Ad5/SIVGag- and Ad5/gp140_jrfl-vaccinated groups were associated with the overall strength of the induced cellular immune responses. To examine the contribution of Mamu-A * 01 allele in vaccine efficacy against SHIV-89.6P challenge, Mamu-A * 01-positive monkeys were immunized with Ad5/SIVGag. Vaccine-mediated protection was significantly more pronounced in the Mamu-A * 01-positive monkeys than in Mamu-A * 01-negative monkeys, suggesting the strong contributions of T-cell epitopes restricted by the Mamu-A * 01 molecule. The implications of these results in the development of an HIV-1 vaccine will be discussed.

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.

Numerous observations have shown that host genetic factors play important roles in disease progression in human immunodeficiency virus type 1 (HIV-1) infection (24, 28). The genetics of the major histocompatibility complex (MHC) appears to be a particularly critical factor (5). Rapid disease progression has been demonstrated to be associated with certain class I HLA molecules, such as the HLA-B35-Cw∗04 haplotype (3, 11). Slow disease progression is linked with several class I HLA molecules, notably HLA-B∗27 and HLA-B∗57 (13, 17, 23). Recently, this protective effect was demonstrated in a mother-to-child HIV-1 transmission setting, where it was found that HLA-B∗27-restricted immunodominant cytotoxic T-lymphocyte (CTL) responses play a crucial role in delaying disease progression (12). Similar protective effects were also observed in simian immunodeficiency virus (SIV) infection, particularly in association with expression of the Mamu-A∗01 MHC allele. For instance, expression of Mamu-A∗01 significantly delayed disease progression in SIVmac 251 infection (8, 9, 25). Since this allele has been consistently associated with the CTL response to an immunodominant epitope derived from the SIV Gag region (SIV Gag residues 181 to 189, designated p11CM), detectable by sensitive tetramer and ELISPOT assays, Mamu-A∗01 monkeys have been favored for evaluation of AIDS vaccine candidates (1, 2, 29). Infection with a pathogenic isolate of simian-human immunodeficiency virus (SHIV 89.6P), causes severe CD4 lymphopenia and rapid disease progression in rhesus monkeys (16, 27). Because of the highly pathogenic nature of this virus and its consistent replication kinetics in rhesus monkeys, this challenge model is widely adopted for evaluation of potential AIDS vaccines (1, 2, 29). To further understand the pathogenesis of SHIV 89.6P infection, particularly the impact of expression of the Mamu-A∗01 allele on disease progression, we monitored a cohort of intravenously infected rhesus monkeys with and without expression of the Mamu-A∗01 allele. Here we report that expression of the Mamu-A∗01 allele was associated with delayed disease progression to AIDS in SHIV 89.6P infection. Early virus-specific CTL responses, especially Mamu-A∗01-restricted CTL responses, likely contributed to the significant reduction of initial viral replication observed in lymphoid tissue. Reduced viral replication in lymphoid tissue allowed lymphoid tissue structure to remain relatively intact, thereby resulting in attenuation of disease progression.

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.