Low-dose M.tb infection but not BCG or MTBVAC vaccination enhances heterologous antibody titres in non-human primates.

Introduction: Mycobacteria are known to exert a range of heterologous effects on the immune system. The mycobacteria-based Freund's Complete Adjuvant is a potent non-specific stimulator of the immune response used in immunization protocols promoting antibody production, and Mycobacterium bovis Bacille Calmette Guérin (BCG) vaccination has been linked with decreased morbidity and mortality beyond the specific protection it provides against tuberculosis (TB) in some populations and age groups. The role of heterologous antibodies in this phenomenon, if any, remains unclear and under-studied. Methods: We set out to evaluate antibody responses to a range of unrelated pathogens following infection with Mycobacterium tuberculosis (M.tb) and vaccination with BCG or a candidate TB vaccine, MTBVAC, in non-human primates. Results: We demonstrate a significant increase in the titer of antibodies against SARS-CoV-2, cytomegalovirus, Epstein-Barr virus, tetanus toxoid, and respiratory syncytial virus antigens following low-dose aerosol infection with M.tb. The magnitude of some of these responses correlated with TB disease severity. However, vaccination with BCG administered by the intradermal, intravenous or aerosol routes, or intradermal delivery of MTBVAC, did not increase antibody responses against unrelated pathogens. Discussion: Our findings suggest that it is unlikely that heterologous antibodies contribute to the non-specific effects of these vaccines. The apparent dysregulation of B cell responses associated with TB disease warrants further investigation, with potential implications for risk of B cell cancers and novel therapeutic strategies.

Broad and potent neutralizing antibodies are elicited in vaccinated individuals following Delta/BA.1 breakthrough infection.

IMPORTANCE: With the emergence of SARS-CoV-2 viral variants, there has been an increase in infections in vaccinated individuals. Here, we isolated monoclonal antibodies (mAbs) from individuals experiencing a breakthrough infection (Delta or BA.1) to determine how exposure to a heterologous Spike broadens the neutralizing antibody response at the monoclonal level. All mAbs isolated had reactivity to the Spike of the vaccine and infection variant. While many mAbs showed reduced neutralization of current circulating variants, we identified mAbs with broad and potent neutralization of BA.2.75.2, XBB, XBB.1.5, and BQ.1.1 indicating the presence of conserved epitopes on Spike. These results indicate that variant-based vaccine boosters have the potential to broaden the vaccine response.

Cross-reactivity of glycan-reactive HIV-1 broadly neutralizing antibodies with parasite glycans.

The HIV-1 Envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs). Env is heavily glycosylated with host-derived N-glycans, and many bnAbs bind to, or are dependent upon, Env glycans for neutralization. Although glycan-binding bnAbs are frequently detected in HIV-infected individuals, attempts to elicit them have been unsuccessful because of the poor immunogenicity of Env N-glycans. Here, we report cross-reactivity of glycan-binding bnAbs with self- and non-self N-glycans and glycoprotein antigens from different life-stages of Schistosoma mansoni. Using the IAVI Protocol C HIV infection cohort, we examine the relationship between S. mansoni seropositivity and development of bnAbs targeting glycan-dependent epitopes. We show that the unmutated common ancestor of the N332/V3-specific bnAb lineage PCDN76, isolated from an HIV-infected donor with S. mansoni seropositivity, binds to S. mansoni cercariae while lacking reactivity to gp120. Overall, these results present a strategy for elicitation of glycan-reactive bnAbs which could be exploited in HIV-1 vaccine development.

Mycobacterium tuberculosis infection is associated with increased B cell responses to unrelated pathogens.

Antigens from Mycobacterium tuberculosis (M.tb), have been shown to stimulate human B cell responses to unrelated recall antigens in vitro. However, it is not known whether natural M.tb infection or whether vaccination with, Mycobacterium bovis BCG, has a similar effect. This study investigated the effects of M.tb infection and BCG vaccination on B cell responses to heterologous pathogen recall antigens. Antibodies against several bacterial and viral pathogens were quantified by ELISA in 68 uninfected controls, 62 individuals with latent TB infection (LTBI) and 107 active pulmonary TB (APTB) cases, and 24 recently BCG-vaccinated adolescents and naive controls. Antibody avidity was investigated using surface plasmon resonance and B cell ELISPOTs were used to measure plasmablast and memory B cell responses (MBC) in APTB cases and healthy donor controls. APTB was associated with higher levels of antibodies to respiratory syncytial virus and measles virus, compared to uninfected controls. BCG vaccination did not alter levels of antibodies against heterologous pathogens. Tetanus toxoid (TT)-specific antibody avidity was increased in APTB cases in comparison to uninfected individuals and the ratio of TT-specific plasmablasts to MBCs in the APTB cases was 7:1. M.tb infection is associated with increased antibody responses to heterologous pathogens in human subjects.

Characterising antibody avidity in individuals of varied Mycobacterium tuberculosis infection status using surface plasmon resonance.

There is increasing evidence supporting a role for antibodies in protection against tuberculosis (TB), with functional antibodies being described in the latent state of TB infection. Antibody avidity is an important determinant of antibody-mediated protection. This study characterised the avidity of antibodies against Ag85A, an immunodominant Mycobacterium tuberculosis (M.tb) antigen and constituent of several anti-TB vaccine candidates, in individuals of varied M.tb infection status. Avidity of Ag85A specific antibodies was measured in 30 uninfected controls, 34 individuals with latent TB infection (LTBI) and 75 active pulmonary TB (APTB) cases, employing the more commonly used chaotrope-based dissociation assays, and surface plasmon resonance (SPR). Chaotrope-based assays indicated that APTB was associated with a higher antibody avidity index compared to uninfected controls [adjusted geometric mean ratio (GMR): 1.641, 95% confidence interval (CI): 1.153, 2.337, p = 0.006, q = 0.018] and to individuals with LTBI [adjusted GMR: 1.604, 95% CI: 1.282, 2.006, p < 0.001, q <0.001]. SPR assays showed that APTB was associated with slower dissociation rates, an indication of higher avidity, compared to uninfected controls (adjusted GMR: 0.796, 95% CI: 0.681, 0.932, p = 0.004, q = 0.012) and there was also weak evidence of more avid antibodies in the LTBI compared to the uninfected controls (adjusted GMR: 0.871, 95% CI: 0.763, 0.994, p = 0.041, q = 0.123). We found no statistically significant differences in anti-Ag85A antibody avidity between the APTB and LTBI groups. This study shows that antibodies of increased avidity are generated against a principle vaccine antigen in M.tb infected individuals. It would be important to determine whether TB vaccines are able to elicit a similar response. Additionally, more research is needed to determine whether antibody avidity is important in protection against infection and disease.

Use of QuantiFERON®-TB Gold in-tube culture supernatants for measurement of antibody responses.

QuantiFERON®-TB Gold in-tube (QFT-GIT) supernatants may be important samples for use in assessment of anti-tuberculosis (TB) antibodies when only limited volumes of blood can be collected and when a combination of antibody and cytokine measurements are required. These analytes, when used together, may also have the potential to differentiate active pulmonary TB (APTB) from latent TB infection (LTBI). However, few studies have explored the use of QFT-GIT supernatants for investigations of antibody responses. This study determined the correlation and agreement between anti-CFP-10 and anti-ESAT-6 antibody concentrations in QFT-GIT nil supernatant and serum pairs from 68 TB household contacts. We also explored the ability of Mycobacterium tuberculosis (M.tb) specific antibodies, or ratios of antibody to interferon gamma (IFN-γ) in QFT-GIT supernatants, to differentiate 97 APTB cases from 58 individuals with LTBI. Sputum smear microscopy was used to define APTB, whereas the QFT-GIT and tuberculin skin test were used to define LTBI. There were strong and statistically significant correlations between anti-CFP-10 and anti-ESAT-6 antibodies in unstimulated QFT-GIT supernatants and sera (r = 0.89; p<0.0001 for both), and no significant differences in antibody concentration between them. Anti-CFP-10 & anti-ESAT-6 antibodies differentiated APTB from LTBI with sensitivities of 88.7% & 71.1% and specificities of 41.4% & 51.7% respectively. Anti-CFP-10 antibody/M.tb specific IFN-γ and anti-ESAT-6 antibody/M.tb specific IFN-γ ratios had sensitivities of 48.5% & 54.6% and specificities of 89.7% and 75.9% respectively. We conclude that QFT-GIT nil supernatants may be used in the place of sera when measuring antibody responses, reducing blood volumes needed for such investigations. Antibodies in QFT-GIT nil supernatants on their own discriminate APTB from LTBI with high sensitivity but have poor specificity, whereas the reverse is true when antibodies are used in combination with M.tb specific cytokines. Further antibody and antibody/cytokine combinations need to be explored to achieve better diagnostic accuracy.

The effect of current Schistosoma mansoni infection on the immunogenicity of a candidate TB vaccine, MVA85A, in BCG-vaccinated adolescents: An open-label trial.

INTRODUCTION: Helminth infection may affect vaccine immunogenicity and efficacy. Adolescents, a target population for tuberculosis booster vaccines, often have a high helminth burden. We investigated effects of Schistosoma mansoni (Sm) on the immunogenicity and safety of MVA85A, a model candidate tuberculosis vaccine, in BCG-vaccinated Ugandan adolescents. METHODS: In this phase II open label trial we enrolled 36 healthy, previously BCG-vaccinated adolescents, 18 with no helminth infection detected, 18 with Sm only. The primary outcome was immunogenicity measured by Ag85A-specific interferon gamma ELISpot assay. Tuberculosis and schistosome-specific responses were also assessed by whole-blood stimulation and multiplex cytokine assay, and by antibody ELISAs. RESULTS: Ag85A-specific cellular responses increased significantly following immunisation but with no differences between the two groups. Sm infection was associated with higher pre-immunisation Ag85A-specific IgG4 but with no change in antibody levels following immunisation. There were no serious adverse events. Most reactogenicity events were of mild or moderate severity and resolved quickly. CONCLUSIONS: The significant Ag85A-specific T cell responses and lack of difference between Sm-infected and uninfected participants is encouraging for tuberculosis vaccine development. The implications of pre-existing Ag85A-specific IgG4 antibodies for protective immunity against tuberculosis among those infected with Sm are not known. MVA85A was safe in this population. TRIAL REGISTRATION: ClinicalTrials.gov NCT02178748.

Humoral Responses to Rv1733c, Rv0081, Rv1735c, and Rv1737c DosR Regulon-Encoded Proteins of Mycobacterium tuberculosis in Individuals with Latent Tuberculosis Infection.

Latent tuberculosis infection (LTBI) is evidence of immunological control of tuberculosis. Dormancy survival regulator (DosR) regulon-encoded proteins may have a role in the maintenance of LTBI. T cell responses to Rv1733c, Rv0081, Rv1735c, and Rv1737c DosR regulon-encoded proteins were found to be most frequent among household contacts of TB cases from Uganda compared to other DosR proteins, but antibody responses were not described. We characterized antibody responses to these proteins in individuals from Uganda. Antibodies to Rv1733c, Rv0081, Rv1735c, and Rv1737c DosR regulon-encoded proteins were measured in 68 uninfected individuals, 62 with LTBI, and 107 with active pulmonary tuberculosis (APTB) cases. There were no differences in the concentrations of antibodies to Rv0081, Rv1735c, and Rv1737c DosR regulon-encoded proteins between individuals with LTBI and APTB and those who were uninfected. LTBI was associated with higher concentrations of antibodies to Rv1733c in female participants [adjusted geometric mean ratio: 1.812, 95% confidence interval (CI): 1.105 2.973, and p = 0.019] but not in males (p value for interaction = 0.060). Antibodies to the four DosR regulon-encoded proteins investigated may not serve as good biomarkers of LTBI in the general population. More of the M.tb proteome needs to be screened to identify proteins that induce strong antibody responses in LTBI.

The Use of Interferon Gamma Inducible Protein 10 as a Potential Biomarker in the Diagnosis of Latent Tuberculosis Infection in Uganda.

BACKGROUND: In the absence of a gold standard for the diagnosis of latent tuberculosis (TB) infection (LTBI), the current tests available for the diagnosis of LTBI are limited by their inability to differentiate between LTBI and active TB disease. We investigated IP-10 as a potential biomarker for LTBI among household contacts exposed to sputum positive active TB cases. METHODS: Active TB cases and contacts were recruited into a cohort with six months' follow-up. Contacts were tested for LTBI using QuantiFERON®-TB Gold In-Tube (QFN) assay and the tuberculin skin test (TST). Baseline supernatants from the QFN assay of 237 contacts and 102 active TB cases were analysed for Mycobacterium tuberculosis (MTB) specific and mitogen specific IP-10 responses. RESULTS: Contacts with LTBI (QFN+TST+) had the highest MTB specific IP-10 responses at baseline, compared to uninfected contacts (QFN-TST-) p<0.0001; and active cases, p = 0.01. Using a cut-off of 8,239 pg/ml, MTB specific IP-10 was able to diagnose LTBI with a sensitivity of 87.1% (95% CI, 76.2-94.3) and specificity of 90.9% (95% CI, 81.3-96.6). MTB specific to mitogen specific IP-10 ratio was higher in HIV negative active TB cases, compared to HIV negative latently infected contacts, p = 0.0004. Concentrations of MTB specific IP-10 were higher in contacts with TST conversion (negative at baseline, positive at 6-months) than in those that were persistently TST negative, p = 0.001. CONCLUSION: IP-10 performed well in differentiating contacts with either latent or active TB from those who were uninfected but was not able to differentiate LTBI from active disease except when MTB specific to mitogen specific ratios were used in HIV negative adults. In addition, IP-10 had the potential to diagnose 'recent TB infection' in persons classified as having LTBI using the TST. Such individuals with strong IP-10 responses would likely benefit from chemoprophylaxis.

Effect of isoniazid preventive therapy on immune responses to mycobacterium tuberculosis: an open label randomised, controlled, exploratory study.

BACKGROUND: With the renewed emphasis to implement isoniazid preventive therapy (IPT) in Sub-Saharan Africa, we investigated the effect of IPT on immunological profiles among household contacts with latent tuberculosis. METHODS: Household contacts of confirmed tuberculosis patients were tested for latent tuberculosis using the QuantiFERON®-TB Gold In-Tube (QFN) assay and tuberculin skin test (TST). HIV negative contacts aged above 5 years, positive to both QFN and TST, were randomly assigned to IPT and monthly visits or monthly visits only. QFN culture supernatants from enrolment and six months' follow-up were analysed for M.tb-specific Th1, Th2, Th17, and regulatory cytokines by Luminex assay, and for M.tb-specific IgG antibody concentrations by ELISA. Effects of IPT were assessed as the net cytokine and antibody production at the end of six months. RESULTS: Sixteen percent of contacts investigated (47/291) were randomised to IPT (n = 24) or no IPT (n = 23). After adjusting for baseline cytokine or antibody responses, and for presence of a BCG scar, IPT (compared to no IPT) resulted in a relative decline in M.tb-specific production of IFN gamma (adjusted mean difference at the end of six months (bootstrap 95% confidence interval (CI), p-value) -1488.6 pg/ml ((-2682.5, -294.8), p = 0.01), and IL- 2 (-213.1 pg/ml (-419.2, -7.0), p = 0.04). A similar decline was found in anti-CFP-10 antibody levels (adjusted geometric mean ratio (bootstrap 95% CI), p-value) 0.58 ((0.35, 0.98), p = 0.04). We found no effect on M.tb-specific Th2 or regulatory or Th17 cytokine responses, or on antibody concentrations to PPD and ESAT-6. CONCLUSIONS: IPT led to a decrease in Th1 cytokine production, and also in the anti CFP-10 antibody concentration. This could be secondary to a reduction in mycobacterial burden or as a possible direct effect of isoniazid induced T cell apoptosis, and may have implications for protective immunity following IPT in tuberculosis-endemic countries. TRIAL REGISTRATION: ISRCTN registry, ISRCTN15705625. Registered on 30(th) September 2015.