Invaplex functions as an intranasal adjuvant for subunit and DNA vaccines co-delivered in the nasal cavity of nonhuman primates.

Development of intranasal vaccines for HIV-1 and other mucosal pathogens has been hampered by the lack of adjuvants that can be given safely to humans. We have found that an intranasal Shigella vaccine (Invaplex) which is well tolerated in humans can also function as an adjuvant for intranasal protein and DNA vaccines in mice. To determine whether Invaplex could potentially adjuvant similar vaccines in humans, we simultaneously administered a simian immunodeficiency virus (SIV) envelope (Env) protein and DNA encoding simian-human immunodeficiency virus (SHIV) with or without Invaplex in the nasal cavity of female rhesus macaques. Animals were intranasally boosted with adenoviral vectors expressing SIV env or gag,pol to evaluate memory responses. Anti-SIV antibodies in sera and nasal, genital tract and rectal secretions were quantitated by ELISA. Intracellular cytokine staining was used to measure Th1-type T cells in blood. Macaques given DNA/protein immunizations with 0.5 mg Invaplex developed greater serum IgG, nasal IgA and cervicovaginal IgA responses to SIV Env and SHIV Gag,Pol proteins when compared to non-adjuvanted controls. Rectal IgA responses to Env were only briefly elevated and not observed to Gag,Pol. Invaplex increased frequencies of IFNγ-producing CD4 and CD8 T cells to the Env protein, but not T cell responses induced by the DNA. Ad-SIV boosting increased Env-specific polyfunctional T cells and Env- and Gag,Pol-specific antibodies in serum and all secretions. The data suggest that Invaplex could be highly effective as an adjuvant for intranasal protein vaccines in humans, especially those intended to prevent infections in the genital or respiratory tract.

Latent simian varicella virus reactivates in monkeys treated with tacrolimus with or without exposure to irradiation.

Simian varicella virus (SVV) infection of primates resembles human varicella-zoster virus (VZV) infection. After primary infection, SVV becomes latent in ganglia and reactivates after immunosuppression or social and environmental stress. Herein, natural SVV infection was established in 5 cynomolgus macaques (cynos) and 10 African green (AG) monkeys. Four cynos were treated with the immunosuppressant tacrolimus (80 to 300 μg/kg/day) for 4 months and 1 was untreated (group 1). Four AG monkeys were exposed to a single dose (200 cGy) of x-irradiation (group 2), and 4 other AG monkeys were irradiated and treated with tacrolimus for 4 months (group 3); the remaining 2 AG monkeys were untreated. Zoster rash developed 1 to 2 weeks after tacrolimus treatment in 3 of 4 monkeys in group 1, 6 weeks after irradiation in 1 of 4 monkeys in group 2, and 1 to 2 weeks after irradiation in all 4 monkeys in group 3. All monkeys were euthanized 1 to 4 months after immunosuppression. SVV antigens were detected immunohistochemically in skin biopsies as well as in lungs of most monkeys. Low copy number SVV DNA was detected in ganglia from all three groups of monkeys, including controls. RNA specific for SVV ORFs 61, 63, and 9 was detected in ganglia from one immunosuppressed monkey in group 1. SVV antigens were detected in multiple ganglia from all immunosuppressed monkeys in every group, but not in controls. These results indicate that tacrolimus treatment produced reactivation in more monkeys than irradiation and tacrolimus and irradiation increased the frequency of SVV reactivation as compared to either treatment alone.

Modification of a common BAL technique to enhance sample diagnostic value.

Bronchoalveolar lavage (BAL) by means of bronchoscopy is a diagnostic tool frequently used for clinical and research purposes in nonhuman primates. Although many institutions use this procedure, the technique is not standardized. One technical aspect that can vary is the method by which fluid is recovered. The purpose of this study was to evaluate differences between 2 different BAL aspiration techniques. Bronchoscopy and BAL fluid collection were performed on 20 rhesus macaques (Macaca mulatta). Data collected for comparison included heart rate, oxygen saturation levels, rectal temperature, volume of fluid collected, total cell count, cell viability, differential cell count, and flow cytometry. Results showed no significant differences in the heart rate, oxygen saturation, or body temperature between the 2 groups. Likewise, differential cell counts and cell viability studies of the retrieved fluid did not differ between methods. Compared with the conventional technique, the modified aspiration technique led to an 8.3% increase in overall fluid yield and a higher concentration of cells recovered. These differences are statistically significant and likely will be clinically relevant in the context of diagnosis.

Tropism-independent protection of macaques against vaginal transmission of three SHIVs by the HIV-1 fusion inhibitor T-1249.

We have assessed the potential of the fusion inhibitory peptide T-1249 for development as a vaginal microbicide to prevent HIV-1 sexual transmission. When formulated as a simple gel, T-1249 provided dose-dependent protection to macaques against high-dose challenge with three different SHIVs that used either CCR5 or CXCR4 for infection (the R5 virus SHIV-162P3, the X4 virus SHIV-KU1 and the R5X4 virus SHIV-89.6P), and it also protected against SIVmac251 (R5). Protection of half of the test animals was estimated by interpolation to occur at T-1249 concentrations of approximately 40-130 muM, whereas complete protection was observed at 0.1-2 mM. In vitro, T-1249 had substantial breadth of activity against HIV-1 strains from multiple genetic subtypes and in a coreceptor-independent manner. Thus, at 1 muM in a peripheral blood mononuclear cell-based replication assay, T-1249 inhibited all 29 R5 viruses, all 12 X4 viruses and all 7 R5X4 viruses in the test panel, irrespective of their genetic subtype. Combining lower concentrations of T-1249 with other entry inhibitors (CMPD-167, BMS-C, or AMD3465) increased the proportion of test viruses that could be blocked. In the PhenoSense assay, T-1249 was active against 636 different HIV-1 Env-pseudotyped viruses of varying tropism and derived from clinical samples, with IC(50) values typically clustered in a 10-fold range approximately 10 nM. Overall, these results support the concept of using T-1249 as a component of an entry inhibitor-based combination microbicide to prevent the sexual transmission of diverse HIV-1 variants.