Defined tuberculosis vaccine, Mtb72F/AS02A, evidence of protection in cynomolgus monkeys.

The development of a vaccine for tuberculosis requires a combination of antigens and adjuvants capable of inducing appropriate and long-lasting T cell immunity. We evaluated Mtb72F formulated in AS02A in the cynomolgus monkey model. The vaccine was immunogenic and caused no adverse reactions. When monkeys were immunized with bacillus Calmette-Guérin (BCG) and then boosted with Mtb72F in AS02A, protection superior to that afforded by using BCG alone was achieved, as measured by clinical parameters, pathology, and survival. We observed long-term survival and evidence of reversal of disease progression in monkeys immunized with the prime-boost regimen. Antigen-specific responses from protected monkeys receiving BCG and Mtb72F/AS02A had a distinctive cytokine profile characterized by an increased ratio between 3 Th1 cytokines, IFN-gamma, TNF, and IL-2 and an innate cytokine, IL-6. To our knowledge, this is an initial report of a vaccine capable of inducing long-term protection against tuberculosis in a nonhuman primate model, as determined by protection against severe disease and death, and by other clinical and histopathological parameters.

Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice.

The Lyme disease vaccine is based on the outer-surface lipoprotein (OspA) of the pathogen Borrelia burgdorferi, and 95% of vaccine recipients develop substantial titers of antibodies against OspA. Here, we identified seven individuals with very low antibody titers after vaccination (low responders). The macrophages of low responders produced less tumor necrosis factor-alpha and interleukin-6 after OspA stimulation and had lower cell-surface expression of Toll-like receptor (TLR) 1 as compared to normal cells, but normal expression of TLR2. TLRs activate innate responses to pathogens, and TLR2 recognizes lipoproteins and peptidoglycan (PGN). After OspA immunization, mice genetically deficient in either TLR2 (TLR2(-/-)) or TLR1 (TLR1(-/-)) produced low titers of antibodies against OspA. Notably, macrophages from TLR2(-/-) mice were unresponsive to OspA and PGN, whereas those from TLR1(-/-) mice responded normally to PGN but not to OspA. These data indicate that TLR1 and TLR2 are required for lipoprotein recognition and that defects in the TLR1/2 signaling pathway may account for human hyporesponsiveness to OspA vaccination.

Tuberculosis vaccine development; from mouse to man.

A tuberculosis (TB) vaccine candidate, Mtb72, was developed following an antigen discovery program involving a combination of expression cloning strategies and evaluation of human immune responses. Adjuvant selection was also performed, resulting in the prioritization of AS02A and AS01B, and an industrial process for vaccine production was developed. Safety, immunogenicity, and protection studies in mice, guinea pigs, rabbits, and monkeys supported the initiation of clinical development of Mtb72f in AS02A.

Evaluation of vaccines in the EU TB Vaccine Cluster using a guinea pig aerosol infection model of tuberculosis.

The TB Vaccine Cluster project funded by the EU Fifth Framework programme aims to provide novel vaccines against tuberculosis that are suitable for evaluation in humans. This paper describes the studies of the protective efficacy of vaccines in a guinea pig aerosol-infection model of primary tuberculosis. The objective was to conduct comparative evaluations of vaccines that had previously demonstrated efficacy in other animal models. Groups of 6 guinea pigs were immunized with vaccines provided by the relevant EU Vaccine Cluster partners. Survival over 17 or 26 weeks was used as the principal measure of vaccine efficacy following aerosol challenge with H37Rv. Counts of mycobacteria in lungs and spleens, and histopathological changes in the lungs, were also used to provide evidence of protection. A total of 24 vaccines were evaluated in 4 experiments each of a different design. A heterologous prime-boost strategy of DNA and MVA, each expressing Ag85A and a fusion protein of ESAT-6 and Ag85B in adjuvant, protected the guinea pigs to the same extent as BCG. Genetically modified BCG vaccines and boosted BCG strategies also protected guinea pigs to the same extent as BCG but not statistically significantly better. A relatively high aerosol-challenge dose and evaluation over a protracted time post-challenge allowed superior protection over BCG to be demonstrated by BCG boosted with MVA and fowl pox vectors expressing Ag85A.

Identification of Mycobacterium tuberculosis vaccine candidates using human CD4+ T-cells expression cloning.

To identify Mycobacterium tuberculosis (Mtb) antigens as candidates for a subunit vaccine against tuberculosis (TB), we have employed a CD4+ T-cell expression screening method. Mtb-specific CD4+ T-cell lines from nine healthy PPD positive donors were stimulated with different antigenic substrates including autologous dendritic cells (DC) infected with Mtb, or cultured with culture filtrate proteins (CFP), and purified protein derivative of Mtb (PPD). These lines were used to screen a genomic Mtb library expressed in Escherichia coli and processed and presented by autologous DC. This screening led to the recovery of numerous T-cell antigens, including both novel and previously described antigens. One of these novel antigens, referred to as Mtb9.8 (Rv0287), was recognized by multiple T-cell lines, stimulated with either Mtb-infected DC or CFP. Using the mouse and guinea pig models of TB, high levels of IFN-gamma were produced, and solid protection from Mtb challenge was observed following immunization with Mtb9.8 formulated in either AS02A or AS01B Adjuvant Systems. These results demonstrate that T-cell screening of the Mtb genome can be used to identify CD4+ T-cell antigens that are candidates for vaccine development.

Identification and characterization of novel recombinant vaccine antigens for immunization against genital Chlamydia trachomatis.

Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection worldwide, with over 91 million cases estimated annually. An effective subunit vaccine against Chlamydia may require a multivalent subunit cocktail of antigens in a single formulation for broad coverage of a heterogeneous major histocompatibility complex population. Herein, we describe the identification of novel C. trachomatis antigens by CD4+ and CD8+ T-cell expression cloning, serological expression cloning, and an in silico analysis of the C. trachomatis genome. These antigens elicited human CD4+ T-cell responses, and a subset proved to be immunogenic and protective when administered as immunoprophylactic vaccines against C. trachomatis challenge. Candidate vaccines consisting of the prioritized C. trachomatis antigens adjuvanted in a GlaxoSmithKline proprietary AS01B adjuvant were prioritized based on induction of solid protection against challenge in C57BL/6 and BALB/c mice with C. trachomatis. Some of the vaccines prevented bacterial shedding and colonization of the upper genital tract to varying degrees by mechanisms that may include CD4+ T cells.

Comparative evaluation of three assays for measurement of dengue virus neutralizing antibodies.

Plaque reduction neutralization tests (PRNTs) are commonly used for measuring levels of dengue virus (DENV) neutralizing antibodies. However, these assays lack a standardized format, generally have a low sample throughput, and are labor-intensive. The objective of the present study was to evaluate two alternative DENV neutralizing antibody assays: an enzyme-linked immunosorbent assay-based microneutralization (MN) assay, and a fluorescent antibody cell sorter-based, DC-SIGN expresser dendritic cell (DC) assay. False-positive rates, serotype specificity, reproducibility, sensitivity, and agreement among the assay methods were assessed using well-characterized but limited numbers of coded test sera. Results showed that all three assays had false-positive rates of less than 10% with titers near the cut-off and generally below the estimated limits of detection. All three methods demonstrated a high degree of specificity and good agreement when used to assay sera and serum mixtures from monovalent vaccinees and sera from patients after primary natural infection, with the only notable exception being moderate-to-high neutralizing antibody titers against DENV 2 measured by PRNT in a mixture containing only DENV 3 and DENV 4 sera. The MN and DC assays demonstrated good reproducibility. All three assays were comparable in their sensitivity, except that the PRNT was less sensitive for measuring DENV 4 antibody, and the MN and DC assays were less sensitive for measuring DENV 2 antibody. However, when used to test sera from persons after tetravalent DENV vaccination or secondary DENV infection, there was poor specificity and poor agreement among the different assays.

Protection of mice and guinea pigs against tuberculosis induced by immunization with a single Mycobacterium tuberculosis recombinant antigen, MTB41.

MTB41 is a Mycobacterium antigen that is recognized by CD4+ T cells early after experimental infection of mice with Mycobacterium tuberculosis and by PBMC from healthy PPD positive individuals. Immunization of mice with plasmid DNA encoding the MTB41 gene sequence results in the development of antigen-specific CD4+ and CD8+ T cells, and protection against challenge with virulent M. tuberculosis. In the present studies, in contrast to DNA immunization, we show, that a strong MTB41-specific CD4+ T cell response, but no MHC class I restricted cytotoxic T lymphocyte (CTL) activity is detected in the spleen cells of infected mice. Therefore, this data suggests that the induction of CD8+ T cell response to MTB41 epitopes by DNA immunization may not be relevant to protection because these epitopes are not recognized during the infectious process. We also compared the repertoire of rMTB41 epitope recognition by CD4+ T cells of M. tuberculosis-infected mice with the recognition repertoire of mice immunized with the recombinant rMTB41 protein. Both regimens of sensitization lead to the recognition of the same molecular epitope. Coincidentally, immunization with the soluble recombinant protein plus adjuvant, a regimen known to generate primarily CD4+ T cells, resulted in induction of protection comparable to BCG in two well-established animal models of tuberculosis (mice and guinea pigs).

Early detection of Borrelia burgdorferi sensu lato infection in Balb/c mice by co-feeding Ixodes ricinus ticks.

In Europe, Borrelia burgdorferi is transmitted by Ixodes ricinus to animals and human. When infected and uninfected ticks co-feed on a host, spirochetes are transmitted from ticks to animal and also to uninfected ticks. Here, we used uninfected ticks to co-feed with infected ticks on mice to evaluate this method to detect early infection in mice. A total of 128 mice were challenged by infected nymphs placed in capsules glued on the back of the mice. Three days later uninfected larvae were added in the capsule to co-feed with infected nymphs and were examined for Borrelia infection after natural detachment. Infection in mice was also determined by xenodiagnosis and by spirochete isolation from ear skin biopsy and back skin biopsy taken at the tick attachment site one month after infection. A total of 111 mice were found to be infected by at least one of these four methods. Borrelia infection was observed in 95% of mice by the co-feeding method, in 92% of mice by xenodiagnosis, in 69% and in 68% of mice by cultivation of ear and back skin biopsies, respectively. Our results demonstrate that the co-feeding method is a very sensitive method which can be used to detect very early infection in mice infected by tick bites.

Assessment of the nasopharyngeal bacterial flora of rhesus macaques: moraxella, Neisseria, haemophilus, and other genera.

The nasopharyngeal bacterial flora of healthy rhesus macaques was surveyed for the presence of Neisseria and Haemophilus species, as well as Moraxella catarrhalis. M. catarrhalis was found both in healthy rhesus macaques and in possibly immunocompromised rhesus macaques. Several Haemophilus spp. that are part of the normal nasopharyngeal bacterial flora of humans were found in many animals; these Haemophilus species included H. parahaemolyticus, H. segnis, and H. parainfluenzae. While Haemophilus influenzae was not identified, it is possible that the identification of H. influenzae types may have been thwarted by the growth of less fastidious species. A number of animals harbored Neisseria spp. such as N. sicca. However, Neisseria meningitidis was not found. In summary, it appears as though the rhesus macaque may be used as a model for M. catarrhalis infections. Moreover, in view of the susceptibility of macaques to organisms of the Haemophilus and Neisseria genera, it is possible that these animals may also accurately model nontypeable H. influenzae and N. meningitidis infections.

The protective effect of the Mycobacterium bovis BCG vaccine is increased by coadministration with the Mycobacterium tuberculosis 72-kilodalton fusion polyprotein Mtb72F in M. tuberculosis-infected guinea pigs.

A tuberculosis vaccine candidate consisting of a 72-kDa polyprotein or fusion protein based upon the Mtb32 and Mtb39 antigens of Mycobacterium tuberculosis and designated Mtb72F was tested for its protective capacity as a potential adjunct to the Mycobacterium bovis BCG vaccine in the mouse and guinea pig models of this disease. Formulation of recombinant Mtb72F (rMtb72F) in an AS02A adjuvant enhanced the Th1 response to BCG in mice but did not further reduce the bacterial load in the lungs after aerosol challenge infection. In the more stringent guinea pig disease model, rMtb72F delivered by coadministration with BCG vaccination significantly improved the survival of these animals compared to BCG alone, with some animals still alive and healthy in their appearance at >100 weeks post-aerosol challenge. A similar trend was observed with guinea pigs in which BCG vaccination was boosted by DNA vaccination, although this increase was not statistically significant due to excellent protection conferred by BCG alone. Histological examination of the lungs of test animals indicated that while BCG controls eventually died from overwhelming lung consolidation, the majority of guinea pigs receiving BCG mixed with rMtb72F or boosted twice with Mtb72F DNA had mostly clear lungs with minimal granulomatous lesions. Lesions were still prominent in guinea pigs receiving BCG and the Mtb72F DNA boost, but there was considerable evidence of lesion healing and airway remodeling and reestablishment. These data support the hypothesis that the coadministration or boosting of BCG vaccination with Mtb72F may limit the lung consolidation seen with BCG alone and may promote lesion resolution and healing. Collectively, these data suggest that enhancing BCG is a valid vaccination strategy for tuberculosis that is worthy of clinical evaluation.

Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein.

Key Ags of Mycobacterium tuberculosis initially identified in the context of host responses in healthy purified protein derivative-positive donors and infected C57BL/6 mice were prioritized for the development of a subunit vaccine against tuberculosis. Our lead construct, Mtb72F, codes for a 72-kDa polyprotein genetically linked in tandem in the linear order Mtb32(C)-Mtb39-Mtb32(N). Immunization of C57BL/6 mice with Mtb72F DNA resulted in the generation of IFN-gamma responses directed against the first two components of the polyprotein and a strong CD8(+) T cell response directed exclusively against Mtb32(C). In contrast, immunization of mice with Mtb72F protein formulated in the adjuvant AS02A resulted in the elicitation of a moderate IFN-gamma response and a weak CD8(+) T cell response to Mtb32c. However, immunization with a formulation of Mtb72F protein in AS01B adjuvant generated a comprehensive and robust immune response, resulting in the elicitation of strong IFN-gamma and Ab responses encompassing all three components of the polyprotein vaccine and a strong CD8(+) response directed against the same Mtb32(C) epitope identified by DNA immunization. All three forms of Mtb72F immunization resulted in the protection of C57BL/6 mice against aerosol challenge with a virulent strain of M. tuberculosis. Most importantly, immunization of guinea pigs with Mtb72F, delivered either as DNA or as a rAg-based vaccine, resulted in prolonged survival (>1 year) after aerosol challenge with virulent M. tuberculosis comparable to bacillus Calmette-Guérin immunization. Mtb72F in AS02A formulation is currently in phase I clinical trial, making it the first recombinant tuberculosis vaccine to be tested in humans.