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.

In vivo inhibition of tryptophan catabolism reorganizes the tuberculoma and augments immune-mediated control of Mycobacterium tuberculosis.

Mycobacterium tuberculosis continues to cause devastating levels of mortality due to tuberculosis (TB). The failure to control TB stems from an incomplete understanding of the highly specialized strategies that M. tuberculosis utilizes to modulate host immunity and thereby persist in host lungs. Here, we show that M. tuberculosis induced the expression of indoleamine 2,3-dioxygenase (IDO), an enzyme involved in tryptophan catabolism, in macrophages and in the lungs of animals (mice and macaque) with active disease. In a macaque model of inhalation TB, suppression of IDO activity reduced bacterial burden, pathology, and clinical signs of TB disease, leading to increased host survival. This increased protection was accompanied by increased lung T cell proliferation, induction of inducible bronchus-associated lymphoid tissue and correlates of bacterial killing, reduced checkpoint signaling, and the relocation of effector T cells to the center of the granulomata. The enhanced killing of M. tuberculosis in macrophages in vivo by CD4+ T cells was also replicated in vitro, in cocultures of macaque macrophages and CD4+ T cells. Collectively, these results suggest that there exists a potential for using IDO inhibition as an effective and clinically relevant host-directed therapy for TB.

Mucosal Vaccination with Heterologous Viral Vectored Vaccine Targeting Subdominant SIV Accessory Antigens Strongly Inhibits Early Viral Replication.

Conventional HIV T cell vaccine strategies have not been successful in containing acute peak viremia, nor in providing long-term control. We immunized rhesus macaques intramuscularly and rectally using a heterologous adenovirus vectored SIV vaccine regimen encoding normally weakly immunogenic tat, vif, rev and vpr antigens fused to the MHC class II associated invariant chain. Immunizations induced broad T cell responses in all vaccinees. Following up to 10 repeated low-dose intrarectal challenges, vaccinees suppressed early viral replication (P=0.01) and prevented the peak viremia in 5/6 animals. Despite consistently undetectable viremia in 2 out of 6 vaccinees, all animals showed evidence of infection induced immune responses indicating that infection had taken place. Vaccinees, with and without detectable viremia better preserved their rectal CD4+ T cell population and had reduced immune hyperactivation as measured by naïve T cell depletion, Ki-67 and PD-1 expression on T cells. These results indicate that vaccination towards SIV accessory antigens vaccine can provide a level of acute control of SIV replication with a suggestion of beneficial immunological consequences in infected animals of unknown long-term significance. In conclusion, our studies demonstrate that a vaccine encoding subdominant antigens not normally associated with virus control can exert a significant impact on acute peak viremia.

Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis.

Tuberculosis (TB) is a global pandaemic, partially due to the failure of vaccination approaches. Novel anti-TB vaccines are therefore urgently required. Here we show that aerosol immunization of macaques with the Mtb mutant in SigH (MtbΔsigH) results in significant recruitment of inducible bronchus-associated lymphoid tissue (iBALT) as well as CD4(+) and CD8(+) T cells expressing activation and proliferation markers to the lungs. Further, the findings indicate that pulmonary vaccination with MtbΔsigH elicited strong central memory CD4(+) and CD8(+) T-cell responses in the lung. Vaccination with MtbΔsigH results in significant protection against a lethal TB challenge, as evidenced by an approximately three log reduction in bacterial burdens, significantly diminished clinical manifestations and granulomatous pathology and characterized by the presence of profound iBALT. This highly protective response is virtually absent in unvaccinated and BCG-vaccinated animals after challenge. These results suggest that future TB vaccine candidates can be developed on the basis of MtbΔsigH.

Aerosol vaccination with AERAS-402 elicits robust cellular immune responses in the lungs of rhesus macaques but fails to protect against high-dose Mycobacterium tuberculosis challenge.

Development of a vaccine against pulmonary tuberculosis may require immunization strategies that induce a high frequency of Ag-specific CD4 and CD8 T cells in the lung. The nonhuman primate model is essential for testing such approaches because it has predictive value for how vaccines elicit responses in humans. In this study, we used an aerosol vaccination strategy to administer AERAS-402, a replication-defective recombinant adenovirus (rAd) type 35 expressing Mycobacterium tuberculosis Ags Ag85A, Ag85B, and TB10.4, in bacillus Calmette-Guérin (BCG)-primed or unprimed rhesus macaques. Immunization with BCG generated low purified protein derivative-specific CD4 T cell responses in blood and bronchoalveolar lavage. In contrast, aerosolized AERAS-402 alone or following BCG induced potent and stable Ag85A/b-specific CD4 and CD8 effector T cells in bronchoalveolar lavage that largely produced IFN-γ, as well as TNF and IL-2. Such responses induced by BCG, AERAS-402, or both failed to confer overall protection following challenge with 275 CFUs M. tuberculosis Erdman, although vaccine-induced responses associated with reduced pathology were observed in some animals. Anamnestic T cell responses to Ag85A/b were not detected in blood of immunized animals after challenge. Overall, our data suggest that a high M. tuberculosis challenge dose may be a critical factor in limiting vaccine efficacy in this model. However, the ability of aerosol rAd immunization to generate potent cellular immunity in the lung suggests that using different or more immunogens, alternative rAd serotypes with enhanced immunogenicity, and a physiological challenge dose may achieve protection against M. tuberculosis.

Granuloma correlates of protection against tuberculosis and mechanisms of immune modulation by Mycobacterium tuberculosis.

BACKGROUND: The BCG vaccine is ineffective against adult tuberculosis. Hence, new antituberculosis vaccines are needed. Correlates of protection against tuberculosis are not known. We studied the effects of BCG vaccination on gene expression in tuberculosis granulomas using macaques. METHODS: Macaques were BCG-vaccinated or sham-vaccinated and then challenged with virulent Mycobacterium tuberculosis. Lung lesions were used for comparative transcriptomics. RESULTS: Vaccinated macaques were protected with lower bacterial burden and immunopathology. Lesions from BCG-vaccinated nonhuman primates (NHPs) showed a better balance of α- and ß-chemokine gene expression with higher levels of ß-chemokine expression relative to nonvaccinated animals. Consistent with this, sham-vaccinated macaques recruited fewer macrophages relative to neutrophils in their lungs. The expression of indoleamine 2,3-dioxygenase (IDO), a known immunosuppressor, was significantly higher in both week 5 and 10 lesions from sham-vaccinated, relative to BCG-vaccinated, NHPs. IDO expression was primarily limited to the nonlymphocytic region of the lesions, within the inner ring structure surrounding the central necrosis. CONCLUSIONS: Our study defines lung gene expression correlates of protective response against tuberculosis, relative to disease, which can potentially be employed to assess the efficacy of candidate antituberculosis vaccines. Mycobacterium tuberculosis may modulate protective immune responses using diverse mechanisms, including increased recruitment of inflammatory neutrophils and the concomitant use of IDO to modulate inflammation.

The Mycobacterium tuberculosis stress response factor SigH is required for bacterial burden as well as immunopathology in primate lungs.

BACKGROUND: Sigma H (sigH) is a major Mycobacterium tuberculosis (Mtb) stress response factor. It is induced in response to heat, oxidative stress, cell wall damage, and hypoxia. Infection of macrophages with the Δ-sigH mutant generates more potent innate immune response than does infection with Mtb. The mutant is attenuated for pathology in mice. METHODS: We used a nonhuman primate (NHP) model of acute tuberculosis, to better understand the phenotype of the Δ-sigH mutant in vivo. NHPs were infected with high doses of Mtb or the mutant, and the progression of tuberculosis was analyzed in both groups using clinical, pathological, microbiological, and immunological parameters. RESULTS: Animals exposed to Mtb rapidly progressed to acute pulmonary tuberculosis as indicated by worsening clinical correlates, high lung bacterial burden, and granulomatous immunopathology. All the animals rapidly succumbed to tuberculosis. On the other hand, the NHPs exposed to the Mtb:Δ-sigH mutant did not exhibit acute tuberculosis, instead showing significantly blunted disease. These NHPs survived the entire duration of the study. CONCLUSIONS: The Mtb:Δ-sigH mutant is completely attenuated for bacterial burden as well as immunopathology in NHPs. SigH and its regulon are required for complete virulence in primates. Further studies are needed to identify the molecular mechanism of this attenuation.

HIV vaccine rationale, design and testing.

A central obstacle to the design of a global HIV vaccine is viral diversity. Antigenic differences in envelope proteins result in distinct HIV serotypes, operationally defined such that antibodies raised against envelope molecules from one serotype will not bind envelope molecules from a different serotype. The existence of serotypes has presented a similar challenge to vaccine development against other pathogens. In such cases, antigenic diversity has been addressed by vaccine design. For example, the poliovirus vaccine includes three serotypes of poliovirus, and Pneumovax presents a cocktail of 23 pneumococcal variants to the immune system. It is likely that a successful vaccine for HIV must also comprise a cocktail of antigens. Here, data relevant to the development of cocktail vaccines, designed to harness diverse, envelope-specific B-cell and T-cell responses, are reviewed.

Spectrum of manifestations of Mycobacterium tuberculosis infection in primates infected with SIV.

To characterize the manifestations of coinfection with M. tuberculosis and SIV infection, we studied 12 SIV-infected rhesus monkeys, six of which were infected intrabronchially with a low dose of Mycobacterium tuberculosis H37Rv. In the six coinfected animals, M. tuberculosis antigen-stimulated lung and blood cells produced high concentrations of IFN-gamma but not IL-4 8-16 weeks after infection. Of the three coinfected animals with high levels of plasma viremia, two developed disseminated tuberculosis and the other died of bacterial peritonitis. Of three coinfected animals with moderate levels of plasma viremia, two had no clinical or radiographic evidence of tuberculosis or progressive SIV infection for 6 months after infection. At neuropsy, pulmonary granulomata were observed and acid-fast organisms or M. tuberculosis were present. These clinical, immunologic and pathologic findings are consistent with those in humans with latent tuberculosis infection (LTBI), and suggest that a model of LTBI in SIV-infected primates can be developed. Such a model will permit delineation of the immunologic and microbial factors that characterize LTBI in HIV-infected persons.

Use of perflubron to enhance lung gene expression: safety and initial efficacy studies in non-human primates.

Use of perflubron (LiquiVent) and other perfluorochemical liquids during intratracheal administration of adenovirus and AAV vectors has been shown to improve total gene expression as well as distribution of expression throughout lungs of spontaneously breathing rodents. To determine if this method could be safely and easily extended to non-human primates, we carried out a pilot investigation in six spontaneously breathing rhesus macaques. Two animals received bronchoscopic administration of recombinant adenovirus vector (type 5 E1-deleted AdCMVlacZ, 4.6 x 10(10) plaque forming units/animal), two animals received vector followed by instillation of perflubron, and two animals received perflubron alone. Instillation of perflubron was well tolerated by the animals and, once recovered from anesthesia, all animals behaved and fed normally until lung harvest. Serial X-rays demonstrated that the perflubron had cleared from lungs of three animals by 48 hours after administration; the fourth animal had a small amount of residual perflubron. Apart from a mild elevation in hepatocellular enzymes, no significant abnormality was noted in complete blood count or serum electrolytes and chemistries. In animals receiving either vector alone or vector with perflubron, in situ beta-galactosidase expression was observed in a variety of cells including large airway, bronchiolar, and alveolar epithelial cells. In summary, use of perflubron was well tolerated in spontaneously breathing macaques. Further studies in larger numbers of animals will help assess the potential efficacy of perflubron for enhancing gene expression and elucidate effects on local and systemic inflammatory responses.