Development and pre-clinical assessment of a 73 kD chimeric fusion protein as a defined sub-unit vaccine for leprosy.

Despite the advances toward the elimination of leprosy through widespread provision of multi-drug therapy to registered patients over the last 2 decades, new case detection rates have stabilized and leprosy remains endemic in a number of localized regions. A vaccine could overcome the inherent limitations of the drug treatment program by providing protection in individuals who are not already harboring the Mycobacterium leprae bacilli at the time of administration and effectively interrupt the transmission cycle over a wider timespan. In this report we present data validating the production of 73f, a chimeric fusion protein incorporating the M. leprae antigens ML2028, ML2346 and ML2044. The 73f protein was recognized by IgG in multibacillary (MB) leprosy patient sera and stimulated IFNγ production within whole blood assays of paucibacillary (PB) leprosy patient and healthy household contacts of MB patients (HHC). When formulated with a TLR4L-containing adjuvant (GLA-SE), 73f stimulated a strong and pluripotent Th1 response that inhibited M. leprae-induced inflammation in mice. We are using these data to develop new vaccine initiatives for the continued and long-term control of leprosy.

Incorporation of immunostimulatory motifs in the transcribed region of a plasmid DNA vaccine enhances Th1 immune responses and therapeutic effect against Mycobacterium tuberculosis in mice.

T-helper type 1 (Th1) immune response is involved in the development of protective immunity against Mycobacterium tuberculosis. Thus, an increase in Th1 and cellular immune responses should lead to enhanced anti-mycobacterial activity. In this study, we aimed to improve Th1 immune responses to a DNA vaccine by adding potentially immunostimulatory nucleotide sequences into the transcribed region downstream of the antigen. The Mycobacterium leprae gene for hsp65, codon-optimized for expression in mammalian cells, was inserted into pVAX1 with and without 3'-sequences containing CpG and dsRNA motifs. When the plasmid contained both motifs, transfected murine macrophage-like RAW264.7 cells showed markedly increased levels of mRNA for immune molecules of Th1 (IFN-α, IL-12) and Th17 (IL-17, IL-23 and IL-6) responses and for T cell co-stimulatory molecules (CD80 and CD86) but not for a Th2 response (IL-4 and IL-10). Immunized mice showed substantially increased serum anti-Hsp65 IgG2a antibody levels and IFN-γ production by spleen cells, confirming enhancement of the Th1 response in vivo. Furthermore, when non-vaccinated mice were infected with H37Rv by low-dose aerosol challenge, and then 4 weeks later were treated with plasmids by intramuscular injection, the mice that had been treated with plasmids containing immunostimulatory motifs showed an enhanced reduction in mycobacterial loads in lung and spleen. We conclude that DNA vaccines may be made more highly immunogenic and more effective for treatment by including transcribed stimulatory sequences.

A subunit vaccine based on biodegradable microspheres carrying rHsp65 protein and KLK protects BALB/c mice against tuberculosis infection.

Of the hundreds of new tuberculosis (TB) vaccine candidates, some have therapeutic value in addition to their prophylactic properties. This is the case for the DNA vaccine encoding heat-shock protein 65 (DNAhsp65) from Mycobacterium leprae. However, there are concerns about the use of DNA vaccines in certain populations such as newborns and pregnant women. Thus, the optimization of vaccination strategies that circumvent this limitation is a priority. This study evaluated the efficacy of a single dose subunit vaccine based on recombinant Hsp65 protein against infection with M. tuberculosis H37Rv. The Hsp65 protein in this study was either associated or not with immunostimulants, and was encapsulated in biodegradable PLGA microspheres. Our results demonstrate that the protein was entrapped in microspheres of adequate diameter to be engulfed by phagocytes. Mice vaccinated with a single dose of Hsp65-microspheres or Hsp65+CpG-microspheres developed both humoral and cellular-specific immune responses. However, they did not protect mice against challenge with M. tuberculosis. By contrast, Hsp65+KLK-microspheres induced specific immune responses that reduced bacilli loads and minimized lung parenchyma damage. These data suggest that a subunit vaccine based on recombinant protein Hsp65 is feasible.

Conformational stability and antibody response to the 18kDa heat-shock protein formulated into different vehicles.

Protein stability is one of the most important obstacles for successful formulation in the development of new-generation vaccines. Here, the 18kDa heat-shock protein (18kDa-hsp) was chemically modified though conjugation with bovine serum albumin or by esterification with N-hydroxysuccinimide ester of palmitic acid. The biologically active conformation of the protein was preserved after chemical modification. The immune responses to the recombinant 18kDa-hsp from Mycobacterium leprae were studied in different presentations: free, copolymerized with bovine serum albumin in aggregates (18kDa-hsp-BSA), and either surface linked to liposomes or entrapped into liposomes. Measuring the antibody production of immunized genetically selected mice has compared the adjuvant effects of liposomes and proteic copolymer. Among the two liposome preparations, the strongest response was obtained with the surface-exposed antigen-liposomes. The copolymer 18kDa-hsp-BSA conferred a high titer of antibody in injected mice, and persisted 70 d after immunization. This approach should prove very useful for designing more effective vaccines by using 18kDa-hsp as carrier protein.

Recombinant BCG as a candidate oral vaccine vector.

Bacille Calmette-Guerin (BCG), currently the most widely used vaccine in the world, was originally administered for many years as an oral vaccine. The low frequency of serious complications, inexpensive production, and adjuvanticity make BCG an ideal candidate for a recombinant vaccine vehicle. Although mycobacteria are slow growing and not yet well characterized genetically, we have recently developed technology for the genetic manipulation of BCG and other mycobacteria. Phage and plasmid systems based on a shuttle strategy to manipulate DNA in Escherichia coli and transfer it to mycobacteria have been developed. We have established that the aminoglycoside phosphotransferase gene can be used as an effective selectable marker in the mycobacteria and that a foreign antigen from Mycobacterium leprae can be expressed in BCG. Furthermore, a thorough analysis of mycobacterial expression sequences has been undertaken to optimize the expression of foreign antigens in BCG. We constructed an expression probe shuttle plasmid with beta-galactosidase as reporter gene, and have used it successfully to identify multiple mycobacteriophage DNA sequences with varying levels of constitutive or regulable promoter activity. Further genetic advances required for development of recombinant BCG into an effective recombinant vaccine vehicle, including possibilities for oral administration, are adumbrated.