Potential determinants of antibody responses after vaccination against SARS-CoV-2 in older persons: the Doetinchem Cohort Study.

BACKGROUND: Immune responses to vaccination vary widely between individuals. The aim of this study was to identify health-related variables potentially underlying the antibody responses to SARS-CoV-2 vaccination in older persons. We recruited participants in the long-running Doetinchem Cohort Study (DCS) who underwent vaccination as part of the national COVID-19 program, and measured antibody concentrations to SARS-CoV-2 Spike protein (S1) and Nucleoprotein (N) at baseline (T0), and a month after both the first vaccination (T1), and the second vaccination (T2). Associations between the antibody concentrations and demographic variables, including age, sex, socio-economic status (SES), comorbidities (cardiovascular diseases and immune mediated diseases), various health parameters (cardiometabolic markers, inflammation markers, kidney- and lung function) and a composite measure of frailty ('frailty index', ranging from 0 to 1) were tested using multivariate models. RESULTS: We included 1457 persons aged 50 to 92 years old. Of these persons 1257 were infection naïve after their primary vaccination series. The majority (N = 954) of these individuals were vaccinated with two doses of BNT162b2 (Pfizer) and their data were used for further analysis. A higher frailty index was associated with lower anti-S1 antibody responses at T1 and T2 for both men (RT1 = -0.095, PT1 = 0.05; RT2 = -0.11, PT2 = 0.02) and women (RT1 = -0.24, PT1 < 0.01; RT2 = -0.15, PT2 < 0.01). After correcting for age and sex the frailty index was also associated with the relative increase in anti-S1 IgG concentrations between the two vaccinations (ß = 1.6, P < 0.01). Within the construct of frailty, history of a cardiac catheterization, diabetes, gastrointestinal disease, a cognitive speed in the lowest decile of the population distribution, and impaired lung function were associated with lower antibody responses after both vaccinations. CONCLUSIONS: Components of frailty play a key role in the primary vaccination response to the BNT162b2 vaccine within an ageing population. Older persons with various comorbidities have a lowered immune response after their first vaccination, and while frail and sick older persons see a stronger increase after their second vaccination compared to healthy people, they still have a lower antibody response after their second vaccination.

Species-Specific Patterns of Gut Metabolic Modules in Dutch Individuals with Different Dietary Habits.

Diet is an important determinant of the human gut microbiome. Here, we analyzed fecal metagenomes of Dutch adults following omnivorous, pescatarian, vegan, and vegetarian diets. We compared the taxonomic composition of individuals from our study with publicly available gut metagenomes from westernized and non-westernized societies. We observed that, despite long-term transition to diets rich in plant fibers (vegan or vegetarian), the microbiomes of these were typical of westernized populations, and similar in composition to omnivores. Although there were no major differences in metabolic modules, we identified differences in the species that contributed to particular functions, such as carbohydrate degradation and short-chain fatty acid metabolism. Overall, this study shows functional redundancy of the microbiomes among westernized populations, which is independent of long-term individual dietary habits. IMPORTANCE Diet is an important modulator of the human gut microbiome, which is susceptible to increased consumption of plant fibers in vegan or vegetarian lifestyles. To investigate this, we compared the gut microbiome of Dutch adults following omnivorous, pescatarian, vegan and vegetarian diets. We did not observe major differences in the gut microbiome composition and function between individuals with different dietary habits. However, we observed differences in the species that contribute to the core functions of the gut microbiome. Our study thus emphasizes the need to better understand the species-specific functional changes associated with dietary habits in the human gut microbiome.

Faecal carriage of Clostridioides difficile is low among veterinary healthcare workers in the Netherlands.

Veterinary healthcare workers are in close contact with many different animals and might be at an increased risk of acquiring Clostridioides difficile. In this cross-sectional study, we assessed the prevalence and risk factors of C. difficile carriage in Dutch veterinary healthcare workers. Participants provided a faecal sample and filled out a questionnaire covering potential risk factors for C. difficile carriage. C. difficile culture positive isolates were polymerase chain reaction (PCR) ribotyped and the presence of toxin genes tcdA, tcdB and cdtA/cdtB was determined. Eleven of 482 [2.3%; 95% confidence interval (CI) 1.3-4.0] veterinary healthcare workers were carriers of C. difficile. Three persons carried C. difficile ribotype 078 (0.6%; 95% CI 0.2-1.8). Risk factors for carriage were health/medication and hygiene related, including poor hand hygiene after patient (animal) contact, and did not include occupational contact with certain animal species. In conclusion, the prevalence of C. difficile carriage in veterinary healthcare workers was low and no indications were found that working in veterinary care is a risk for C. difficile carriage.

Impact of long-term dietary habits on the human gut resistome in the Dutch population.

The human gut microbiome plays a central role in health and disease. Environmental factors, such as lifestyle and diet, are known to shape the gut microbiome as well as the reservoir of resistance genes that these microbes harbour; the resistome. In this study we assessed whether long-term dietary habits within a single geographical region (the Netherlands) impact the human gut resistome. Faecal samples from Dutch omnivores, pescatarians, vegetarians and vegans were analysed by metagenomic shotgun sequencing (MSS) (n = 149) and resistome capture sequencing approach (ResCap) (n = 64). Among all diet groups, 119 and 145 unique antibiotic resistance genes (ARGs) were detected by MSS or ResCap, respectively. Five or fifteen ARGs were shared between all diet groups, based on MSS and ResCap, respectively. The total number of detected ARGs by MSS or ResCap was not significantly different between the groups. MSS also revealed that vegans have a distinct microbiome composition, compared to other diet groups. Vegans had a lower abundance of Streptococcus thermophilus and Lactococcus lactis compared to pescatarians and a lower abundance of S. thermophilus when compared to omnivores. In summary, our study showed that long-term dietary habits are not associated with a specific resistome signature.

ESBL/pAmpC-producing Escherichia coli and Klebsiella pneumoniae carriage among veterinary healthcare workers in the Netherlands.

BACKGROUND: Animals are a reservoir for ESBL/pAmpC-producing Escherichia coli/Klebsiella pneumoniae (ESBL-E/K). We investigated the association between occupational contact with different types of animals and the prevalence of ESBL-E/K carriage among veterinary healthcare workers, assessed molecular characteristics of ESBL-E/K, and followed-up on the ESBL-E/K carriage status of participants and their household members. METHODS: Participants completed a questionnaire about their contact with animals at work and at home, health status, travel behaviour and hygiene, and sent in a faecal sample which was tested for the presence of ESBL-E/K. Resistance genes were typed using PCR and sequencing. ESBL-E/K positive participants and their household members were followed up after 6 months. Risk factors were analysed using multivariable logistic regression methods. RESULTS: The prevalence of ESBL-E/K carriage was 9.8% (47/482; 95%CI 7.4-12.7). The most frequently occurring ESBL genes were blaCTX-M-15, blaCTX-M-14 and blaDHA-1. The predominant sequence type was ST131. None of the occupation related factors, such as contact with specific animal species, were significantly associated with ESBL-E/K carriage, whereas travel to Africa, Asia or Latin America in the past 6 months (OR 4.4), and stomach/bowel complaints in the past 4 weeks (OR 2.2) were. Sixteen of 33 initially ESBL-E/K positive participants (48.5%) tested positive again 6 months later, in 14 persons the same ESBL gene and E. coli ST was found. Four of 23 (17.4%) household members carried ESBL-E/K, in three persons this was the same ESBL gene and E. coli ST as in the veterinary healthcare worker. CONCLUSIONS: Despite the absence of specific occupation related risk factors, ESBL-E/K carriage in veterinary healthcare workers was high compared to the prevalence in the general Dutch population (5%). This indicates that occupational contact with animals is a potential source of ESBL-E/K for the population at large.

Do vegetarians less frequently carry ESBL/pAmpC-producing Escherichia coli/Klebsiella pneumoniae compared with non-vegetarians?

BACKGROUND: ESBL and plasmid-mediated AmpC (pAmpC)-producing Enterobacteriaceae are frequently found on meat products in Dutch retail, especially on poultry. OBJECTIVES: We investigated whether vegetarians are at lower risk of carrying ESBL/pAmpC-producing Escherichia coli/Klebsiella pneumoniae (ESBL-E/K) compared with persons who consume meat. METHODS: Vegetarians, pescatarians (vegetarians who eat fish) and non-vegetarians (persons who eat meat at least three times per week) were asked to send in a faecal sample and a questionnaire. ESBL-E/K were cultured and MLSTs were determined. ESBL/pAmpC genes were analysed using PCR and sequencing. The risk of ESBL-E/K carriage in the three study groups was analysed using multivariable logistic regression. RESULTS: Prevalence of ESBL-E/K carriage was 8.0% in vegetarians (63/785; 95% CI 6.3-10.1), 6.9% in pescatarians (27/392; 95% CI 4.8-9.8) and 3.8% in non-vegetarians (14/365; 95% CI 2.3-6.3). Multivariable analysis showed an OR for ESBL-E/K carriage of 2.2 for vegetarians (95% CI 1.2-4.0) and 1.6 for pescatarians (95% CI 0.8-3.2) compared with non-vegetarians. The predominant MLST was E. coli ST131 and the most common ESBL genes were blaCTX-M-15, blaCTX-M-27, blaCTX-M-14 and blaCTX-M-1 in all diet groups. Independent risk factors for ESBL-E/K carriage were travel to Africa/Latin America/Asia (OR 4.6; 95% CI 2.8-7.7) in the past 6 months and rarely/never washing hands before food preparation (OR 2.5; 95% CI 1.2-5.0). CONCLUSIONS: Vegetarians and pescatarians did not have a lower risk of ESBL-E/K carriage compared with non-vegetarians, indicating that eating meat is not an important risk factor for ESBL-E/K carriage.

Viral evolution in HLA-B27-restricted CTL epitopes in human immunodeficiency virus type 1-infected individuals.

The HLA-B27 allele is over-represented among human immunodeficiency virus type 1-infected long-term non-progressors. In these patients, strong CTL responses targeting HLA-B27-restricted viral epitopes have been associated with long-term asymptomatic survival. Indeed, loss of control of viraemia in HLA-B27 patients has been associated with CTL escape at position 264 in the immunodominant KK10 epitope. This CTL escape mutation in the viral Gag protein has been associated with severe viral attenuation and may require the presence of compensatory mutations before emerging. Here, we studied sequence evolution within HLA-B27-restricted CTL epitopes in the viral Gag protein during the course of infection of seven HLA-B27-positive patients. Longitudinal gag sequences obtained at different time points around the time of AIDS diagnosis were obtained and analysed for the presence of mutations in epitopes restricted by HLA-B27, and for potential compensatory mutations. Sequence variations were observed in the HLA-B27-restricted CTL epitopes IK9 and DR11, and the immunodominant KK10 epitope. However, the presence of sequence variations in the HLA-B27-restricted CTL epitopes could not be associated with an increase in viraemia in the majority of the patients studied. Furthermore, we observed low genetic diversity in the gag region of the viral variants throughout the course of infection, which is indicative of low viral replication and corresponds to the low viral load observed in the HLA-B27-positive patients. These data indicated that control of viral replication can be maintained in HLA-B27-positive patients despite the emergence of viral mutations in HLA-B27-restricted epitopes.

Low level of HIV-1 evolution after transmission from mother to child.

Mother-to-child HIV-1 transmission pairs represent a good opportunity to study the dynamics of CTL escape and reversion after transmission in the light of shared and non-shared HLA-alleles. Mothers share half of their HLA alleles with their children, while the other half is inherited from the father and is generally discordant between mother and child. This implies that HIV-1 transmitted from mother to child enters a host environment to which it has already partially adapted. Here, we studied viral evolution and the dynamics of CTL escape mutations and reversion of these mutations after transmission in the context of shared and non-shared HLA alleles in viral variants obtained from five mother-to-child transmission pairs. Only limited HIV-1 evolution was observed in the children after mother-to-child transmission. Viral evolution was mainly driven by forward mutations located inside CTL epitopes restricted by HLA alleles inherited from the father, which may be indicative of CTL pressure.

HIV-1 replication fitness of HLA-B*57/58:01 CTL escape variants is restored by the accumulation of compensatory mutations in gag.

Expression of HLA-B*57 and the closely related HLA-B*58:01 are associated with prolonged survival after HIV-1 infection. However, large differences in disease course are observed among HLA-B*57/58:01 patients. Escape mutations in CTL epitopes restricted by these HLA alleles come at a fitness cost and particularly the T242N mutation in the TW10 CTL epitope in Gag has been demonstrated to decrease the viral replication capacity. Additional mutations within or flanking this CTL epitope can partially restore replication fitness of CTL escape variants. Five HLA-B*57/58:01 progressors and 5 HLA-B*57/58:01 long-term nonprogressors (LTNPs) were followed longitudinally and we studied which compensatory mutations were involved in the restoration of the viral fitness of variants that escaped from HLA-B*57/58:01-restricted CTL pressure. The Sequence Harmony algorithm was used to detect homology in amino acid composition by comparing longitudinal Gag sequences obtained from HIV-1 patients positive and negative for HLA-B*57/58:01 and from HLA-B*57/58:01 progressors and LTNPs. Although virus isolates from HLA-B*57/58:01 individuals contained multiple CTL escape mutations, these escape mutations were not associated with disease progression. In sequences from HLA-B*57/58:01 progressors, 5 additional mutations in Gag were observed: S126N, L215T, H219Q, M228I and N252H. The combination of these mutations restored the replication fitness of CTL escape HIV-1 variants. Furthermore, we observed a positive correlation between the number of escape and compensatory mutations in Gag and the replication fitness of biological HIV-1 variants isolated from HLA-B*57/58:01 patients, suggesting that the replication fitness of HLA-B*57/58:01 escape variants is restored by accumulation of compensatory mutations.

The presence of CXCR4-using HIV-1 prior to start of antiretroviral therapy is an independent predictor of delayed viral suppression.

The emergence of CXCR4-using HIV variants (X4-HIV) is associated with accelerated disease progression in the absence of antiretroviral therapy. However, the effect of X4-HIV variants on the treatment response remains unclear. Here we determined whether the presence of X4-HIV variants influenced the time to undetectable viral load and CD4+ T cell reconstitution after initiation of cART in 732 patients. The presence of X4-HIV variants was determined by MT-2 assay prior to cART initiation and viral load and CD4+ T cell counts were analyzed every 3 to 6 months during a three year follow-up period. Kaplan-Meier and Cox proportional hazard analyses were performed to compare time to viral suppression and the absolute CD4+ T cell counts and increases in CD4+ T cell counts during follow-up were compared for patients with and without X4-HIV at start of cART. Patients harboring X4-HIV variants at baseline showed a delay in time to achieve viral suppression below the viral load detection limit. This delay in viral suppression was independently associated with high viral load and the presence of X4-HIV variants. Furthermore, the absolute CD4+ T cell counts were significantly lower in patients harboring X4-HIV variants at all time points during follow-up. However, no differences were observed in the increase in absolute CD4+ T cell numbers after treatment initiation, indicating that the reconstitution of CD4+ T cells is independent of the presence of X4-HIV variants. The emergence of X4-HIV has been associated with an accelerated CD4+ T cell decline during the natural course of infection and therefore, patients who develop X4-HIV variants may benefit from earlier treatment initiation in order to obtain faster reconstitution of the CD4+ T cell population to normal levels.

Gag sequence variation in a human immunodeficiency virus type 1 transmission cluster influences viral replication fitness.

Three men from a proven homosexual human immunodeficiency virus type 1 (HIV-1) transmission cluster showed large variation in their clinical course of infection. To evaluate the effect of evolution of the same viral variant in these three patients, we analysed sequence variation in the capsid protein and determined the impact of the observed variation on viral replication fitness in vitro. Viral gag sequences from all three patients contained a mutation at position 242, T242N or T242S, which have been associated with lower virus replication in vitro. Interestingly, HIV-1 variants from patients with a progressive clinical course of infection developed compensatory mutations within the capsid that restored viral fitness, instead of reversion of the T242S mutation. In HIV-1 variants from patient 1, an HLA-B57(+) elite controller, no compensatory mutations emerged during follow-up.

HIV-1 envelope diversity 1 year after seroconversion predicts subsequent disease progression.

OBJECTIVE: Recent studies have suggested that the dynamics of HIV-1 evolutionary rate reflect the rate of disease progression. We wished to determine whether viral diversity early in infection is predictive of the subsequent disease course. DESIGN: HIV-1 envelope diversity at seroconversion and 1 year thereafter from 89 homosexual participants of the Amsterdam Cohort Studies on HIV infection and AIDS was correlated with clinical endpoints and markers of disease progression. METHODS: Heteroduplex mobility assay (HMA) and sequencing followed by calculation of pairwise genetic distances were applied to determine HIV-1 envelope diversity. The HMA pattern (presence or absence of heteroduplexes) and sequence diversity were each tested for correlation with the clinical course of infection. RESULTS: HMA pattern at 1-year postseroconversion was significantly associated with progression to AIDS and AIDS-related death, with presence of heteroduplexes associated with accelerated disease progression. Moreover, not only this dichotomous measure of viral diversity (absence or presence of heteroduplexes), but also genetic diversity itself was associated with disease course. HMA pattern was an independent predictor of accelerated disease progression, also when CCR5 genotype, human leukocyte antigen (HLA)-type, viral load, CD4 T-cell counts, and coreceptor use at viral load set point were included in the analysis. CONCLUSION: Viral diversity early in HIV-1 infection is predictive of the subsequent disease progression. It remains to be established whether viral diversity itself plays a causal role in the increased damage to the immune system or whether it is a reflection of immune pressure or other selective forces.