Dihydrolipoamide dehydrogenase-Lpd (Rv0462)-specific T cell recall responses are higher in healthy household contacts of TB: a novel immunodominant antigen from M. tuberculosis

Abstract The partial effectiveness against pulmonary tuberculosis (PTB), displayed by the existing tuberculosis (TB) vaccine, bacillus Calmette-Guérin (BCG), highlights the need for novel vaccines to replace or improve BCG. In TB immunology, antigen-specific cellular immune response is frequently considered indispensable. Latency-associated antigens are intriguing as targets for TB vaccine development. The mycobacterial protein, dihydrolipoamide dehydrogenase (Lpd; Rv0462), the third enzyme of the pyruvate dehydrogenase (PDH) complex, facilitates Mycobacterium tuberculosis to resist host reactive nitrogen intermediates. Multicolor flow cytometry analysis of whole-blood cultures showed higher Lpd-specific Th1 recall response (IFN-γ, TNF-α, and IL-2; P = 0.0006) and memory CD4+ and CD8+ T cells (CCR7+ CD45RA− and CCR7− CD45RA−) in healthy household contacts (HHC) of TB (P &amp;lt; 0.0001), which is comparable with or higher than the standard antigens, ESAT-6 and CFP-10. The frequency of Lpd-specific multifunctional T cells was higher in HHC compared with PTB patients. However, there is no significant statistical correlation. Regulatory T cell (Treg) analysis of HHCs and active TB patients demonstrated very low Lpd-specific CD4+ Tregs relative to ESAT-6 and CFP-10. Our study demonstrates that the Lpd antigen induces a strong cellular immune response in healthy mycobacteria-infected individuals. In consideration of this population having demonstrated immunologic protection against active TB disease development, our data are encouraging about the possible use of Lpd as a target for further TB subunit vaccine development.</jats:p

Proteomics Analysis of Three Different Strains of Mycobacterium tuberculosis under In vitro Hypoxia and Evaluation of Hypoxia Associated Antigen’s Specific Memory T Cells in Healthy Household Contacts

In vitro mimicking conditions are thought to reflect the environment experienced by M. tuberculosis inside the host granuloma. The majority of the in vitro dormancy experimental models used laboratory adapted strain H37Rv or Erdman strain over the prevalent clinical strains involved during disease outbreaks. Thus, we included the most prevalent clinical strains (S7 and S10) of M. tuberculosis from south India in addition to H37Rv for our in vitro oxygen depletion (hypoxia) experimental model. Cytosolic proteins were prepared from the hypoxic cultures, resolved by two-dimensional electrophoresis (2-DE) and protein spots were characterized by mass spectrometry. Totally 49 spots were characterized as over-expressed or newly appeared between the 3 strains. Two antigens (ESAT-6, Lpd) out of the 49 characterized spots were readily available in recombinant form in our lab. Hence, these 2 genes were overexpressed, purified and used for in vitro stimulation of whole blood collected from healthy household contacts (HHC) and active pulmonary tuberculosis patients (PTB). Multicolour flow cytometry analysis showed high levels of antigen specific CD4+ central memory T cells in circulation of HHC when compared to PTB (p<0.005 for ESAT-6 and p<0.0005 for Lpd). This shows proteins that are predicted to be upregulated during in vitro hypoxia in most prevalent clinical strains would bring the possible potential immunogens. In vitro hypoxia experiments with most prevalent clinical strains would also bring the probable true representative antigens that involved during adaption mechanism

The influence of reduced oxygen availability on gene expression in laboratory (H37Rv) and clinical strains (S7 and S10) of Mycobacterium tuberculosis

Mycobacterium tuberculosis has the ability to persist within the host in a dormant stage. One important condition believed to contribute to dormancy is reduced access to oxygen known as hypoxia. However, the response of M. tuberculosis to such hypoxia condition is not fully characterized. Virtually all dormant models against tuberculosis tested in animals used laboratory strain H37Rv or Erdman strain. But major outbreaks of tuberculosis (TB) occur with the strains that have widely different genotypes and phenotypes compared to H37Rv. In this study, we used a custom oligonucleotide microarray to determine the overall transcriptional response of laboratory strain (H37Rv) and most prevalent clinical strains (S7 and S10) of M. tuberculosis from South India to hypoxia. Analysis of microarray results revealed that a total of 1161 genes were differentially regulated (>= 1.5 fold change) in H37Rv, among them 659 genes upregulated and 502 genes down regulated. Microarray data of clinical isolates showed that a total of 790 genes were differentially regulated in S7 among which 453 genes were upregulated and 337 down regulated. Interestingly, numerous genes were also differentially regulated in S10 (total 2805 genes) of which 1463 genes upregulated and 1342 genes down regulated during reduced oxygen condition (Wayne's model). One hundred and thirty-four genes were found common and upregulated among all three strains (H37Rv, S7, and S10) and can be targeted for drug/vaccine development against TB. (C) 2015 Published by Elsevier B.V

Treatment with native heterodimeric IL-15 increases cytotoxiclymphocytes and reduces SHIV RNA in lymph nodes

B cell follicles in secondary lymphoid tissues represent an immune privileged sanctuary for AIDS viruses, in part because cytotoxic CD8(+) T cells are mostly excluded from entering the follicles that harbor infected T follicular helper (T-FH) cells. We studied the effects of native heterodimeric IL-15 (hetIL-15) treatment on uninfected rhesus macaques and on macaques that had spontaneously controlled SHIV infection to low levels of chronic viremia. hetIL-15 increased effector CD8(+) T lymphocytes with high granzyme B content in blood, mucosal sites and lymph nodes, including virus-specific MHC-peptide tetramer+ CD8(+) cells in LN. Following hetIL-15 treatment, multiplexed quantitative image analysis (histo-cytometry) of LN revealed increased numbers of granzyme B+ T cells in B cell follicles and SHIV RNA was decreased in plasma and in LN. Based on these properties, hetIL-15 shows promise as a potential component in combination immunotherapy regimens to target AIDS virus sanctuaries and reduce long-term viral reservoirs in HIV-1 infected individuals

Low Spike Antibody Levels and Impaired BA.4/5 Neutralization in Patients with Multiple Myeloma or Waldenstrom’s Macroglobulinemia after BNT162b2 Booster Vaccination

Patients with symptomatic monoclonal gammopathies have impaired humoral responses to COVID-19 vaccination. Their ability to recognize SARS-CoV-2 Omicron variants is of concern. We compared the response to BNT162b2 mRNA vaccinations of patients with multiple myeloma (MM, n = 60) or Waldenstrom’s macroglobulinemia (WM, n = 20) with healthy vaccine recipients (n = 37). Patient cohorts on active therapy affecting B cell development had impaired binding and neutralizing antibody (NAb) response rate and magnitude, including several patients lacking responses, even after a 3rd vaccine dose, whereas non-B cell depleting therapies had a lesser effect. In contrast, MM and WM cohorts off-therapy showed increased NAb with a broad response range. ELISA Spike-Receptor Binding Domain (RBD) Ab titers in healthy vaccine recipients and patient cohorts were good predictors of the ability to neutralize not only the original WA1 but also the most divergent Omicron variants BA.4/5. Compared to WA1, significantly lower NAb responses to BA.4/5 were found in all patient cohorts on-therapy. In contrast, the MM and WM cohorts off-therapy showed a higher probability to neutralize BA.4/5 after the 3rd vaccination. Overall, the boost in NAb after the 3rd dose suggests that repeat vaccination of MM and WM patients is beneficial even under active therapy