Yves Lobet

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-α 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.

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 Mtb32C-Mtb39-Mtb32N. Immunization of C57BL/6 mice with Mtb72F DNA resulted in the generation of IFN-γ responses directed against the first two components of the polyprotein and a strong CD8+ T cell response directed exclusively against Mtb32C. In contrast, immunization of mice with Mtb72F protein formulated in the adjuvant AS02A resulted in the elicitation of a moderate IFN-γ 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-γ and Ab responses encompassing all three components of the polyprotein vaccine and a strong CD8+ response directed against the same Mtb32C 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.

Attachment of Borrelia burgdorferi within Ixodes scapularis mediated by outer surface protein A

Borrelia burgdorferi outer surface protein (Osp) A has been used as a Lyme disease vaccine that blocks transmission: OspA antibodies of immune hosts enter ticks during blood feeding and destroy spirochetes before transmission to the host can occur. B. burgdorferi produce OspA in the gut of unfed Ixodes scapularis ticks, and many spirochetes repress OspA production during the feeding process. This preferential expression suggests that OspA may have an important function in the vector. Here we show that OspA mediates spirochete attachment to the tick gut by binding to an I. scapularis protein. The binding domains reside in the central region and COOH-terminus of OspA. OspA also binds to itself, suggesting that spirochete-spirochete interactions may further facilitate adherence in the gut. OspA-mediated attachment in the tick provides a possible mechanism for how stage-specific protein expression can contribute to pathogenesis during the B. burgdorferi natural cycle.

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

ABSTRACT 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.

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-γ, 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.

Developing a nontypeable Haemophilus influenzae (NTHi) vaccine

There is a current high demand for nontypable Haemophilus influenzae (NTHi) vaccines. Various options for the composition of such vaccines are possible. Decisions about the vaccine composition have to take into account the antigenic variability of NTHi, so even complex immunogens such as whole bacteria would preferentially have a tailor-made antigenic composition. We will present a summary of NTHi vaccine development, describing research efforts from SmithKline Beecham and other laboratories. Currently, major (P1, P2, P4, P5) and minor (P6, D15, TbpA/B, ellipsis) outer membrane proteins, LPS, adhesins (HMW, Hia, pili, P5) are being studied. Preclinical results with LPD, P5 (LB1) and OMP26 from our laboratories will be described including the use of animal models of otitis and lung infection.

Inhibition of Borrelia burgdorferi-Tick Interactions In Vivo by Outer Surface Protein A Antibody

Borrelia burgdorferi outer surface protein (Osp) A is preferentially expressed by spirochetes in the Ixodes scapularis gut and facilitates pathogen-vector adherence in vitro. Here we examined B. burgdorferi-tick interactions in vivo by using Abs directed against OspA from each of the three major B. burgdorferi sensu lato genospecies: B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii. Abs directed against B. burgdorferi sensu stricto (isolate N40) destroy the spirochete and can protect mice from infection. In contrast, antisera raised against OspA from B. afzelii (isolate ACA-1) and B. garinii (isolate ZQ-1) bind to B. burgdorferi N40 but are not borreliacidal against the N40 isolate. Our present studies assess whether these selected OspA Abs interfere with B. burgdorferi-tick attachment in a murine model of Lyme disease with I. scapularis. We examined engorged ticks that had fed on B. burgdorferi N40-infected scid mice previously treated with OspA (N40, ACA-1, ZQ-1, or mAb C3.78) or control Abs. OspA-N40 antisera or mAb C3.78 destroyed B. burgdorferi N40 within the engorged ticks. In contrast, treatment of mice with OspA-ACA-1 and OspA-ZQ-1 antisera did not kill B. burgdorferi N40 within the ticks but did effectively interfere with B. burgdorferi-I. scapularis adherence, thereby preventing efficient colonization of the vector. These studies show that nonborreliacidal OspA Abs can inhibit B. burgdorferi attachment to the tick gut, highlighting the importance of OspA in spirochete-arthropod interactions in vivo.

Prospects for a better vaccine against tuberculosis

There have been many new promising approaches to developing human vaccines against tuberculosis (TB). Advances in gene and antigen identification, availability of genome sequences, a greater understanding of immune mechanisms in resistance to TB, the development of adjuvants and delivery systems to stimulate T-cell immunity, and increased funding from public and private agencies are some of the reasons for progress in this area. Dozens of vaccine candidates have been tested in animal models in recent years, and several of these are poised to move into clinical trials in the next several years. Thus, there is renewed optimism for the potential of developing new and improved TB vaccines.

Transmission of Borrelia garinii OspA serotype 4 to BALB/c mice by Ixodes ricinus ticks collected in the field.

ABSTRACT In Europe, Borrelia garinii OspA serotype 4 has been isolated from the cerebrospinal fluid of patients but, up to now, has never been identified among culture isolates from Ixodes ricinus ticks. This information raises the question of whether OspA serotype 4 is transmitted by I. ricinus in nature. In the present study, I. ricinus nymphs collected in an area of endemicity in southern Germany were allowed to feed on mice. Cultivation of ear biopsy specimens showed that six of seven B. garinii-infected mice were infected by OspA serotype 4. In contrast, very few B. garinii OspA serotype 4 organisms were isolated directly from the ticks which infected the mice; most isolates were B. afzelii. The infected mice transmitted mainly OspA serotype 4 to xenodiagnostic ticks, preferentially in combination with B. afzelii.

The Lyme disease vaccine candidate outer surface protein A (OspA) in a formulation compatible with human use protects mice against natural tick transmission of B. burgdorferi

Development of a vaccine for the Lyme disease spirochete, Borrelia burgdorferi, has focused on the bacterial lipoprotein, major outer surface protein A (OspA). With few exceptions, testing of OspA vaccines in animal models has involved challenge with needle inoculation of cultured spirochetes. Recombinant OspA proteins from two OspA divergent strains of B. burgdorferi were tested for their vaccine potential in three different strains of mice challenged with laboratory reared ticks with a high rate of B. burgdorferi infection. All formulations of the B. burgdorferi sensu stricto derived OspA vaccine protected all strains of mice when challenged by ticks infected with an OspA homologous strain of the spirochete, whereas heterologous OspA from B. afzelii did not protect. Furthermore, ticks feeding on protected mice had reduced OspA levels compared to unvaccinated controls.