Valerie A. Reese

The Importance of Adjuvant Formulation in the Development of a Tuberculosis Vaccine

An effective protein-based vaccine for tuberculosis will require a safe and effective adjuvant. There are few adjuvants in approved human vaccines, including alum and the oil-in-water–based emulsions MF59 (Novartis Vaccines and Diagnostics), AS03 and AS04 (GlaxoSmithKline Biologics), AF03 (Sanofi), and liposomes (Crucell). When used with pure, defined proteins, both alum and emulsion adjuvants are effective at inducing primarily humoral responses. One of the newest adjuvants in approved products is AS04, which combines monophosphoryl lipid A, a TLR-4 agonist, with alum. In this study, we compared two adjuvants: a stable oil-in-water emulsion (SE) and a stable oil-in-water emulsion incorporating glucopyranosyl lipid adjuvant, a synthetic TLR-4 agonist (GLA-SE), each together with a recombinant protein, ID93. Both the emulsion SE and GLA-SE adjuvants induce potent cellular responses in combination with ID93 in mice. ID93/SE induced Th2-biased immune responses, whereas ID93/GLA-SE induced multifunctional CD4+ Th1 cell responses (IFN-γ, TNF-α, and IL-2). The ID93/GLA-SE vaccine candidate induced significant protection in mice and guinea pigs, whereas no protection was observed with ID93/SE, as assessed by reductions in bacterial burden, survival, and pathology. These results highlight the importance of properly formulating subunit vaccines with effective adjuvants for use against tuberculosis.

Therapeutic immunization against Mycobacterium tuberculosis is an effective adjunct to antibiotic treatment.

BACKGROUND Recent advances in rational adjuvant design and antigen selection have enabled a new generation of vaccines with potential to treat and prevent infectious disease. The aim of this study was to assess whether therapeutic immunization could impact the course of Mycobacterium tuberculosis infection with use of a candidate tuberculosis vaccine antigen, ID93, formulated in a synthetic nanoemulsion adjuvant, GLA-SE, administered in combination with existing first-line chemotherapeutics rifampicin and isoniazid. METHODS We used a mouse model of fatal tuberculosis and the established cynomolgus monkey model to design an immuno-chemotherapeutic strategy to increase long-term survival and reduce bacterial burden, compared with standard antibiotic chemotherapy alone. RESULTS This combined approach induced robust and durable pluripotent antigen-specific T helper-1-type immune responses, decreased bacterial burden, reduced the duration of conventional chemotherapy required for survival, and decreased M. tuberculosis-induced lung pathology, compared with chemotherapy alone. CONCLUSIONS These results demonstrate the ability of therapeutic immunization to significantly enhance the efficacy of chemotherapy against tuberculosis and other infectious diseases, with implications for treatment duration, patient compliance, and more optimal resource allocation.

Chitin microneedles for an easy-to-use tuberculosis skin test.

An easy-to-use tuberculosis skin test is developed with chitin microneedles that deliver purified protein derivative at the correct skin depth and result in a positive test in BCG-immunized guinea pigs.

Immune Subdominant Antigens as Vaccine Candidates against Mycobacterium tuberculosis

Unlike most pathogens, many of the immunodominant epitopes from Mycobacterium tuberculosis are under purifying selection. This startling finding suggests that M. tuberculosis may gain an evolutionary advantage by focusing the human immune response against selected proteins. Although the implications of this to vaccine development are incompletely understood, it has been suggested that inducing strong Th1 responses against Ags that are only weakly recognized during natural infection may circumvent this evasion strategy and increase vaccine efficacy. To test the hypothesis that subdominant and/or weak M. tuberculosis Ags are viable vaccine candidates and to avoid complications because of differential immunodominance hierarchies in humans and experimental animals, we defined the immunodominance hierarchy of 84 recombinant M. tuberculosis proteins in experimentally infected mice. We then combined a subset of these dominant or subdominant Ags with a Th1 augmenting adjuvant, glucopyranosyl lipid adjuvant in stable emulsion, to assess their immunogenicity in M. tuberculosis–naive animals and protective efficacy as measured by a reduction in lung M. tuberculosis burden of infected animals after prophylactic vaccination. We observed little correlation between immunodominance during primary M. tuberculosis infection and vaccine efficacy, confirming the hypothesis that subdominant and weakly antigenic M. tuberculosis proteins are viable vaccine candidates. Finally, we developed two fusion proteins based on strongly protective subdominant fusion proteins. When paired with the glucopyranosyl lipid adjuvant in stable emulsion, these fusion proteins elicited robust Th1 responses and limited pulmonary M. tuberculosis for at least 6 wk postinfection with a single immunization. These findings expand the potential pool of M. tuberculosis proteins that can be considered as vaccine Ag candidates.

Mucosal delivery switches the response to an adjuvanted tuberculosis vaccine from systemic TH1 to tissue-resident TH17 responses without impacting the protective efficacy

Pulmonary tuberculosis (TB) remains one of the leading causes of infectious disease death despite widespread usage of the BCG vaccine. A number of new TB vaccines have moved into clinical evaluation to replace or boost the BCG vaccine including ID93+GLA-SE, an adjuvanted subunit vaccine. The vast majority of new TB vaccines in trials are delivered parenterally even though intranasal delivery can augment lung-resident immunity and protective efficacy in small animal models. Parenteral immunization with the adjuvanted subunit vaccine ID93+GLA-SE elicits robust TH1 immunity and protection against aerosolized Mycobacterium tuberculosis in mice and guinea pigs. Here we describe the immunogenicity and efficacy of this vaccine when delivered intranasally. Intranasal delivery switches the CD4 T cell response from a TH1 to a TH17 dominated tissue-resident response with increased frequencies of ID93-specific cells in both the lung tissue and at the lung surface. Surprisingly these changes do not affect the protective efficacy of ID93+GLA-SE. Unlike intramuscular immunization, ID93+GLA does not require the squalene-based oil-in-water emulsion SE to elicit protective CD4 T cells when delivered intranasally. Finally we demonstrate that TNF and the IL-17 receptor are dispensable for the efficacy of the intranasal vaccine suggesting an alternative mechanism of protection.

Mycobacterium tuberculosis Rv0198c, a putative matrix metalloprotease is involved in pathogenicity

We are interested in the role of proteases in the biology of the global human pathogen, Mycobacterium tuberculosis. We have focused on a putative matrix metalloprotease, Rv0198c. In order to investigate its role we constructed an unmarked chromosomal deletion of the gene and analysed the phenotype of the resulting mutant. No differences in growth in axenic culture were seen and there was no measurable change in overall protease activity in cell-free extracts. Transcriptome analysis revealed a small number of changes in gene expression in aerobic growth, with Rv2488c and Rv1971 being over 40-fold up-regulated and qor (Rv1454c) being 20-fold down-regulated; in addition, changes were seen in members of the heat shock regulon. Virulence assays demonstrated that the mutant was able to replicate in human macrophage-like cells (THP-1 cell line) to a comparable degree with the wild-type. However, the mutant was hyper-virulent in the SCID and C57BL/6 mouse models. Our data suggest that Rv0198c plays a role during infection.

Interferon γ and Tumor Necrosis Factor Are Not Essential Parameters of CD4+ T-Cell Responses for Vaccine Control of Tuberculosis

BACKGROUND Mycobacterium tuberculosis infects one third of the worlds population and causes >8 million cases of tuberculosis annually. New vaccines are necessary to control the spread of tuberculosis. T cells, interferon γ (IFN-γ), and tumor necrosis factor (TNF) are necessary to control M. tuberculosis infection in both humans and unvaccinated experimental animal models. However, the immune responses necessary for vaccine efficacy against M. tuberculosis have not been defined. The multifunctional activity of T-helper type 1 (TH1) cells that simultaneously produce IFN-γ and TNF has been proposed as a candidate mechanism of vaccine efficacy. METHODS We used a mouse model of T-cell transfer and aerosolized M. tuberculosis infection to assess the contributions of TNF, IFN-γ, and inducible nitric oxide synthase (iNOS) to vaccine efficacy. RESULTS CD4(+) T cells were necessary and sufficient to transfer protection against aerosolized M. tuberculosis, but neither CD4(+) T cell-produced TNF nor host cell responsiveness to IFN-γ were necessary. Transfer of Tnf(-/-) CD4(+) T cells from vaccinated donors to Ifngr(-/-) recipients was also sufficient to confer protection. Activation of iNOS to produce reactive nitrogen species was not necessary for vaccine efficacy. CONCLUSIONS Induction of TH1 cells that coexpress IFN-γ and TNF is not a requirement for vaccine efficacy against M. tuberculosis, despite these cytokines being essential for control of M. tuberculosis in nonvaccinated animals.

Protection and long-lived immunity induced by the ID93/GLA-SE vaccine candidate against a clinical Mycobacterium tuberculosis isolate

ABSTRACT Mycobacterium tuberculosis HN878 represents a virulent clinical strain from the W-Beijing family, which has been tested in small animal models in order to study its virulence and its induction of host immune responses following infection. This isolate causes death and extensive lung pathology in infected C57BL/6 mice, whereas lab-adapted strains, such as M. tuberculosis H37Rv, do not. The use of this clinically relevant isolate of M. tuberculosis increases the possibilities of assessing the long-lived efficacy of tuberculosis vaccines in a relatively inexpensive small animal model. This model will also allow for the use of knockout mouse strains to critically examine key immunological factors responsible for long-lived, vaccine-induced immunity in addition to vaccine-mediated prevention of pulmonary immunopathology. In this study, we show that the ID93/glucopyranosyl lipid adjuvant (GLA)-stable emulsion (SE) tuberculosis vaccine candidate, currently in human clinical trials, is able to elicit protection against M. tuberculosis HN878 by reducing the bacterial burden in the lung and spleen and by preventing the extensive lung pathology induced by this pathogen in C57BL/6 mice.

Broadened immunity and protective responses with emulsion-adjuvanted H5 COBRA-VLP vaccines

A number of challenges for developing a protective pre-pandemic influenza A vaccine exists including predicting the target influenza strain and designing the vaccine for an immunologically naïve population. Manufacturing and supply of the vaccine would also require implementing ways to increase coverage for the largest number of people through dose-sparing methods, while not compromising the potency of the vaccine. Previously, our group described a novel hemagglutinin (HA) for H5N1 influenza derived from a methodology termed computationally optimized broadly reactive antigen (COBRA). This report describes a strategy combining a COBRA-based HA vaccine with an oil-in-water emulsion, resulting in a dose-sparing, immunologically broadened, and protective response against multiple H5N1 isolates. Here, we show that an emulsion-based adjuvant enhances the magnitude and breadth of antibody responses with both a wild-type H5HA (H5N1 WT) and the H5N1 COBRA HA VLP vaccines. The H5N1 COBRA HA VLP, combined with an emulsion adjuvant, elicited HAI specific antibodies against a larger panel of H5N1 viruses that resulted in protection against challenge as efficiently as the homologous, matched vaccine.

The ID93 Tuberculosis Vaccine Candidate Does Not Induce Sensitivity to Purified Protein Derivative

ABSTRACT The tuberculin skin test (TST) is a simple and inexpensive test to determine whether individuals have been exposed to Mycobacterium tuberculosis. This test is not always reliable, however, in people previously immunized with BCG and/or who have been exposed to environmental mycobacterial species due to a reaction to purified protein derivative (PPD) used in the skin test. An issue with BCG, therefore, is that the resulting sensitization to PPD in some individuals compromises the diagnostic use of the skin test. The ability to induce protective immune responses without sensitizing to the tuberculin skin test will be important properties of next-generation tuberculosis (TB) vaccine candidates. We show here that guinea pigs immunized with the candidate TB vaccine ID93/GLA-SE, currently in clinical trials, do not react to intradermal PPD administration. In contrast, positive DTH responses to both ID93 and components thereof were induced in ID93/GLA-SE-immunized animals, indicating robust but specific cellular responses were present in the immunized animals. Noninterference with the TST is an important factor for consideration in the development of a vaccine against M. tuberculosis.