C57BL/6 mice were vaccinated with plasmid DNA encoding Ag85 from Mycobacterium tuberculosis, with Ag85 protein in adjuvant, or with a combined DNA prime-protein boost regimen. While DNA immunization, as previously described, induced robust Th1-type cytokine responses, protein-in-adjuvant vaccination elicited very poor cytokine responses, which were 10-fold lower than those observed with DNA immunization alone. Injection of Ag85 DNA-primed mice with 30 to 100 g of purified Ag85 protein in adjuvant increased the interleukin-2 and gamma interferon (IFN-␥) response in spleen two-to fourfold. Further, intracellular cytokine analysis by flow cytometry also showed an increase in IFN-␥-producing CD4 ؉ T cells in DNA-primedprotein-boosted animals, compared to those that received only the DNA vaccination. Moreover, these responses appeared to be better sustained over time. Antibodies were readily produced by all three methods of immunization but were exclusively of the immunoglobulin G1 (IgG1) isotype following protein immunization in adjuvant and preferentially of the IgG2a isotype following DNA and DNA prime-protein boost vaccination. Finally, protein boosting increased the protective efficacy of the DNA vaccine against an intravenous M. tuberculosis H37Rv challenge infection, as measured by CFU or relative light unit counts in lungs 1 and 2 months after infection. The capacity of exogenously given protein to boost the DNA-primed vaccination effect underlines the dominant role of Th1-type CD4؉ helper T cells in mediating protection.Tuberculosis (TB) remains a major health problem affecting millions of people worldwide. The only TB vaccine presently available is an attenuated strain of Mycobacterium bovis termed M. bovis BCG. The efficacy of BCG remains controversial, particularly against pulmonary TB in young adults (5), and development of a better vaccine is urgently needed to counter the global threat of this disease (22).Secreted and surface-exposed cell wall proteins are major antigens recognized by the protective immune response against TB and immunization with whole-culture filtrate, a rich source of these extracellular proteins, can protect mice and guinea pigs to some extent against subsequent challenge with the tubercle bacillus (1, 14, 15). A major portion of the secreted proteins in Mycobacterium tuberculosis and BCG culture filtrate is formed by the Ag85 complex, a 30-to 32-kDa family of three proteins (Ag85A, Ag85B, and Ag85C) (38) which all possess a mycoloyltransferase enzyme activity required for the biogenesis of cord factor (4), a dominant structure necessary for maintaining cell wall integrity (19,29). Ag85 complex induces strong T-cell proliferation and gamma interferon (IFN-␥) production in most healthy individuals infected with M. tuberculosis and/or Mycobacterium leprae (24) and in BCGvaccinated mice (16), making it a promising candidate as a protective antigen. Vaccination with naked plasmid DNA encoding Ag85A and Ag85B can stimulate strong humoral and cell-mediated immune responses and confer si...

For efficient prevention of viral infections and cross protection, simultaneous targeting of multiple viral epitopes is a powerful strategy. Llama heavy chain antibody fragments (VHH) against the trimeric envelope proteins of Respiratory Syncytial Virus (Fusion protein), Rabies virus (Glycoprotein) and H5N1 Influenza (Hemagglutinin 5) were selected from llama derived immune libraries by phage display. Neutralizing VHH recognizing different epitopes in the receptor binding sites on the spikes with affinities in the low nanomolar range were identified for all the three viruses by viral neutralization assays. By fusion of VHH with variable linker lengths, multimeric constructs were made that improved neutralization potencies up to 4,000-fold for RSV, 1,500-fold for Rabies virus and 75-fold for Influenza H5N1. The potencies of the VHH constructs were similar or better than best performing monoclonal antibodies. The cross protection capacity against different viral strains was also improved for all three viruses, both by multivalent (two or three identical VHH) and biparatopic (two different VHH) constructs. By combining a VHH neutralizing RSV subtype A, but not subtype B with a poorly neutralizing VHH with high affinity for subtype B, a biparatopic construct was made with low nanomolar neutralizing potency against both subtypes. Trivalent anti-H5N1 VHH neutralized both Influenza H5N1 clade1 and 2 in a pseudotype assay and was very potent in neutralizing the NIBRG-14 Influenza H5N1 strain with IC50 of 9 picomolar. Bivalent and biparatopic constructs against Rabies virus cross neutralized both 10 different Genotype 1 strains and Genotype 5.The results show that multimerization of VHH fragments targeting multiple epitopes on a viral trimeric spike protein is a powerful tool for anti-viral therapy to achieve “best-in-class” and broader neutralization capacity.

BALB/c (H-2d ) and C57BL/6 (H-2 b ) mice were infected intravenously with Mycobacterium tuberculosis H37Rv or vaccinated intramuscularly with plasmid DNA encoding each of the three mycolyl transferases Ag85A, Ag85B, and Ag85C from M. tuberculosis. Th1-type spleen cell cytokine secretion of interleukin-2 (IL-2) and gamma interferon (IFN-␥) was analyzed in response to purified Ag85 components and synthetic overlapping peptides covering the three mature sequences. Tuberculosis-infected C57BL/6 mice reacted strongly to some peptides from Ag85A and Ag85B but not from Ag85C, whereas tuberculosis-infected BALB/c mice reacted only to peptides from Ag85A. In contrast, spleen cells from both mouse strains produced elevated levels of IL-2 and IFN-␥ following vaccination with Ag85A, Ag85B, and Ag85C DNA in response to peptides of the three Ag85 proteins, and the epitope repertoire was broader than in infected mice. Despite pronounced sequence homology, a number of immunodominant regions contained component specific epitopes. Thus, BALB/c mice vaccinated with all three Ag85 genes reacted against the same amino acid region, 101 to 120, that was also immunodominant for Ag85A in M. bovis BCG-vaccinated and tuberculosis-infected H-2 d haplotype mice, but responses were completely component specific. In C57BL/6 mice, a cross-reactive T-cell response was detected against two carboxy-terminal peptides spanning amino acids 241 to 260 and 261 to 280 of Ag85A and Ag85B. These regions were not recognized at all in C57BL/6 mice vaccinated with Ag85C DNA. Our results underline the need for comparative analysis of all three Ag85 components in future vaccination studies.

Johne's disease, or paratuberculosis, is a chronic granulomatous enteritis in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) affecting principally cattle, sheep and goats. Primarily, there are two clinical signs: cachexia and chronic diarrhea (less common in goats and sheep). This disease results in considerable economic losses in livestock industry, particularly the dairy sector. The route of transmission is mostly by the fecal-oral route, but hygienic measures and culling of shedding animals are not sufficient to eradicate this disease. Moreover, diagnostic tools available at this moment are not powerful enough to perform early and specific diagnosis. Existing vaccines, based on whole killed or live-attenuated bacteria, can delay the onset of clinical symptoms but do not protect against infection. Moreover, vaccinated animals develop antibodies that interfere with existing serodiagnostic tests for paratuberculosis and they become reactive in the tuberculin skin test, used for the control of bovine tuberculosis. This review summarizes the current knowledge of the immune responses induced by MAP infection, with focus on cattle studies. It provides an overview of the existing MAP vaccines and comments on the development of second-generation subunit vaccines based on new technologies.