Aneesh Thakur

Immune markers and correlates of protection for vaccine induced immune responses.

Vaccines have been a major innovation in the history of mankind and still have the potential to address the challenges posed by chronic intracellular infections including tuberculosis, HIV and malaria which are leading causes of high morbidity and mortality across the world. Markers of an appropriate humoral response currently remain the best validated correlates of protective immunity after vaccination. Despite advancements in the field of immunology over the past few decades currently there are, however, no sufficiently validated immune correlates of vaccine induced protection against chronic infections in neither human nor veterinary medicine. Technological and conceptual advancements within cell-mediated immunology have led to a number of new immunological read-outs with the potential to emerge as correlates of vaccine induced protection. For T(H)1 type responses, antigen-specific production of interferon-gamma (IFN-γ) has been promoted as a quantitative marker of protective cell-mediated immune responses over the past couple of decades. More recently, however, evidence from several infections has pointed towards the quality of the immune response, measured through increased levels of antigen-specific polyfunctional T cells capable of producing a triad of relevant cytokines, as a better correlate of sustained protective immunity against this type of infections. Also the possibilities to measure antigen-specific cytotoxic T cells (CTL) during infection or in response to vaccination, through recombinant major histocompatibility complex (MHC) class I tetramers loaded with relevant peptides, has opened a new vista to include CTL responses in the evaluation of protective immune responses. Here, we review different immune markers and new candidates for correlates of a protective vaccine induced immune response against chronic infections and how successful they have been in defining the protective immunity in human and veterinary medicine.

Detection of Mycobacterium bovis and Mycobacterium tuberculosis from Cattle: Possible Public Health Relevance

Mycobacterium bovis and Mycobacterium tuberculosis infect both animals and humans. The disease epidemiology by these agents differs in developed and developing countries due to the differences in the implementation of the prevention and control strategies. The present study describes the detection of M. bovis and M. tuberculosis from specimens of lungs and pulmonary lymph nodes of four cattle died in an organized herd of 183 cattle in the state of Himachal Pradesh, India, with inconclusive skin test results. Identification and distinction of these closely related mycobacterial species was done by PCR-RFLP targeting hsp65 gene followed by spacer oligonucleotide typing. Mixed infection of M. bovis and M. tuberculosis was detected in one cattle.

Cell-Mediated and Humoral Immune Responses after Immunization of Calves with a Recombinant Multiantigenic Mycobacterium avium subsp. paratuberculosis Subunit Vaccine at Different Ages

ABSTRACT Neonates and juvenile ruminants are very susceptible to paratuberculosis infection. This is likely due to a high degree of exposure from their dams and an immature immune system. To test the influence of age on vaccine-induced responses, a cocktail of recombinant Mycobacterium avium subsp. paratuberculosis proteins (MAP0217, MAP1508, MAP3701c, MAP3783, and MAP1609c/Ag85B) was formulated in a cationic liposome adjuvant (CAF01) and used to vaccinate animals of different ages. Male jersey calves were divided into three groups that were vaccinated at 2, 8, or 16 weeks of age and boosted twice at weeks 4 and 12 relative to the first vaccination. Vaccine-induced immune responses, the gamma interferon (IFN-γ) cytokine secretion and antibody responses, were followed for 20 weeks. In general, the specific responses were significantly elevated in all three vaccination groups after the first booster vaccination with no or only a minor effect from the second booster. However, significant differences were observed in the immunogenicity levels of the different proteins, and it appears that the older age group produced a more consistent IFN-γ response. In contrast, the humoral immune response is seemingly independent of vaccination age as we found no difference in the IgG1 responses when we compared the three vaccination groups. Combined, our results suggest that an appropriate age of vaccination should be considered in vaccination protocols and that there is a possible interference of vaccine-induced immune responses with weaning (week 8).

Characterization of the long-term immune response to vaccination against Mycobacterium avium subsp. paratuberculosis in Danish dairy cows

Vaccination of cattle against Mycobacterium avium subsp. paratuberculosis (MAP) provides partial protection by delayed shedding of MAP and reduced numbers of clinically affected animals. The duration of vaccine induced immune response is not known. The primary objective of this study was therefore to characterize the long-term effect of whole-cell based vaccination against MAP on the immune response. A secondary objective was to evaluate whether immunodiagnosis of MAP and Mycobacterium bovis infections is affected by MAP vaccination. Two studies were performed: (1) A retrospective longitudinal study including 895 vaccinated and 2526 non-vaccinated dairy cows in 9 Danish dairy herds aiming at characterizing the long-term antibody-response to vaccination; and (2) a cross-sectional study of responses in the IFN-γ assay carried out in 140 vaccinated animals in two herds to evaluate the effect of vaccination on the cell-mediated immune response and to evaluate a possible interference with the diagnosis of M. bovis infections. The results showed that 37% of samples from vaccinated animals and 5% of samples from non-vaccinated animals, respectively, were test positive in the milk antibody ELISA. The prevalence of antibody responses of the vaccinated animals was relatively constant from 2 to 6 years of age, but decreased in older animals. Among the 140 vaccinated animals 88% tested positive with the IFN-γ test to johnin PPD and 50% responded to PPDb with IFN-γ production above a similar cut-off. Although Denmark is free of M. bovis, two of the vaccinated animals responded with higher IFN-γ levels when cultured with PPDb compared to PPDa. In conclusion, immunization with whole-cell MAP vaccines elicits both humoral and cell-mediated immune reactions, which may interfere with surveillance and diagnosis of both MAP and M. bovis infections using currently available tests.

Increasing the ex vivo antigen-specific IFN-γ production in subpopulations of T cells and NKp46+ cells by anti-CD28, anti-CD49d and recombinant IL-12 costimulation in cattle vaccinated with recombinant proteins from Mycobacterium avium subspecies paratuberculosis

T cells, which encounter specific antigen (Ag), require additional signals to mount a functional immune response. Here, we demonstrate activation of signal 2, by anti-CD28 mAb (aCD28) and other costimulatory molecules (aCD49d, aCD5), and signal 3, by recombinant IL-12, enhance Ag-specific IFN-γ secretion by CD4, CD8, γδ T cells and NK cells. Age matched male jersey calves, experimentally infected with Mycobacterium avium subsp. paratuberculosis (MAP), were vaccinated with a cocktail of recombinant MAP proteins or left unvaccinated. Vaccine induced ex vivo recall responses were measured through Ag-specific IFN-γ production by ELISA and flow cytometry. There was a significant increase in production of IFN-γ by T cell subsets or NKp46+ cells cultured in the presence of Ag and aCD28/aCD49d. The increase was accompanied by an increase in the integrated median fluorescence intensity (iMFI) of activated T cells. Addition of rIL-12 induced a significant additive effect leading to a maximum increase in responder frequency of Ag-specific T cell subsets or NKp46+ cells with a heavy bias toward IFN-γ production by CD4 T cells. We provide the first description of using aCD28/aCD49d costimulation to potentiate an Ag-specific increase in the production of IFN-γ in bovine immunology. The study also shows the degree of signaling in T cells is regulated by the costimulatory environment.

Targeting the Mincle and TLR3 receptor using the dual agonist cationic adjuvant formulation 9 (CAF09) induces humoral and polyfunctional memory T cell responses in calves

There is a need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis and Mycobacterium avium subsp. paratuberculosis in a number of species. One of the main challenges for vaccine development is the lack of safe adjuvants that induce protective immune responses. Cationic Adjuvant Formulation 01 (CAF01)—an adjuvant based on trehalose dibehenate (TDB) and targeting the Mincle receptor—has entered human trials based on promising pre-clinical results in a number of species. However, in cattle CAF01 only induces weak systemic immune responses. In this study, we tested the ability of three pattern recognition receptors, either alone or in combination, to activate bovine monocytes and macrophages. We found that addition of the TLR3 agonist, polyinosinic:polycytidylic acid (Poly(I:C)) to either one of the Mincle receptor agonists, TDB or monomycoloyl glycerol (MMG), enhanced monocyte activation, and calves vaccinated with CAF09 containing MMG and Poly(I:C) had increased cell-mediated and humoral immune response compared to CAF01 vaccinated animals. In contrast to the highly reactogenic Montanide ISA 61 VG, CAF09-primed T cells maintained a higher frequency of polyfunctional CD4+ T cells (IFN-γ+ TNF-α+ IL-2+). In conclusion, CAF09 supports the development of antibodies along with a high-quality cell-mediated immune response and is a promising alternative to oil-in-water adjuvant in cattle and other ruminants.

Seroprevalence of viral and bacterial diseases among the bovines in Himachal Pradesh, India

Aim: The study was designed to measure the seroprevalence of viral and bacterial diseases: Infectious bovine rhinotracheitis, bovine viral diarrhea, bovine leukemia, bovine parainfluenza, bovine respiratory syncytial disease, brucellosis, and paratuberculosis among bovine of Himachal Pradesh during the year 2013-2015. Materials and Methods: The serum samples were collected from seven districts of state, namely, Bilaspur, Kangra, Kinnaur, Lahul and Spiti, Mandi, Sirmour, and Solan. The samples were screened using indirect ELISA kits to measure the seroprevalence of viral and bacterial diseases. Results: The overall seroprevalence of infectious bovine rhinotracheitis was 24.24%, bovine viral diarrhea 1.52%, bovine leukemia 9.09%, bovine parainfluenza 57.58%, bovine respiratory syncytial disease 50%, brucellosis 19.69%, and paratuberculosis 9.09% in Himachal Pradesh. The seroprevalence of bovine rhinotracheitis, bovine leukemia, bovine parainfluenza, bovine respiratory syncytial disease, and paratuberculosis in the state varied significantly (p<0.01) while was insignificant for bovine viral diarrhea and brucellosis (p>0.01). Multiple seropositivity has been observed in this study. Bovine parainfluenza virus 3 was observed commonly in mixed infection with almost all viruses and bacteria under study. Conclusion: The viral and bacterial diseases are prevalent in the seven districts of Himachal Pradesh investigated in the study. Therefore, appropriate management practices and routine vaccination programs should be adopted to reduce the prevalence of these diseases.