Next-generation sequencing (NGS) has instigated the research on the role of the microbiome in health and disease. The compositional nature of such microbiome datasets makes it however challenging to identify those microbial taxa that are truly associated with an intervention or health outcome. Quantitative microbiome profiling overcomes the compositional structure of microbiome sequencing data by integrating absolute quantification of microbial abundances into the NGS data. Both cell-based methods (e.g. flow cytometry) and molecular methods (qPCR) have been used to determine the absolute microbial abundances, but to what extend different quantification methods generate similar quantitative microbiome profiles has so far not been explored. Here we compared relative microbiome profiling (without incorporation of microbial quantification) to three variations of quantitative microbiome profiling: 1) microbial cell counting using flow cytometry (QMP); 2) counting of microbial cells using flow cytometry combined with Propidium Monoazide pre-treatment of fecal samples before metagenomics DNA isolation in order to only profile the microbial composition of intact cells (QMP-PMA), and; 3) molecular based quantification of the microbial load using qPCR targeting the 16S rRNA gene. Although qPCR and flow cytometry both resulted in accurate and strongly correlated results when quantifying the bacterial abundance of a mock community of bacterial cells, the two methods resulted in highly divergent quantitative microbial profiles when analyzing the microbial composition of fecal samples from 16 healthy volunteers. These differences could not be attributed to the presence of free extracellular prokaryotic DNA in the fecal samples as sample pre-treatment with Propidium Monoazide did not improve the concordance between qPCR-based and flow cytometry-based QMP. Also lack of precision of qPCR was ruled out as a major cause of the disconcordant findings, since quantification of the fecal microbial load by the highly sensitive digital droplet PCR correlated strongly with qPCR. In conclusion, quantitative microbiome profiling is an elegant approach to bypass the compositional nature of microbiome NGS data, however it is important to realize that technical sources of variability may introduce substantial additional bias depending on the quantification method being used.
Abstract Background The urgent need for, but limited availability of, SARS-CoV-2 vaccines worldwide has led to widespread consideration of dose sparing strategies, particularly single vaccine dosing of individuals with prior SARS-CoV-2 infection. Methods We evaluated SARS-CoV-2 specific antibody responses following a single-dose of BNT162b2 (Pfizer-BioNTech) mRNA vaccine in 155 previously SARS-CoV-2-infected individuals participating in a population-based prospective cohort study of COVID-19 patients. Participants varied widely in age, comorbidities, COVID-19 severity and time since infection, ranging from 1 to 15 months. Serum antibody titers were determined at time of vaccination and one week after vaccination. Responses were compared to those in SARS-CoV-2-naive health care workers after two BNT162b2 mRNA vaccine doses. Results Within one week of vaccination, IgG antibody levels to virus spike and RBD proteins increased 27 to 29-fold and neutralizing antibody titers increased 12-fold, exceeding titers of fully vaccinated SARS-CoV-2-naive controls (95% credible interval (CrI): 0.56 to 0.67 v. control 95% CrI: −0.16 to −0.02). Pre-vaccination neutralizing antibody titers had the largest positive mean effect size on titers following vaccination (95% CrI (0.16 to 0.45)). COVID-19 severity, the presence of comorbidities and the time interval between infection and vaccination had no discernible impact on vaccine response. Conclusion A single dose of BNT162b2 mRNA vaccine up to 15 months after SARS-CoV-2 infection provides neutralizing titers exceeding two vaccine doses in previously uninfected individuals. These findings support wide implementation of a single-dose mRNA vaccine strategy after prior SARS-CoV-2 infection.
Current SARS-CoV-2 vaccines are losing efficacy against emerging variants and may not protect against future novel coronavirus outbreaks, emphasizing the need for more broadly protective vaccines. To inform the development of a pan-coronavirus vaccine, we investigated the presence and specificity of cross-reactive antibodies against the spike (S) proteins of human coronaviruses (hCoV) after SARS-CoV-2 infection and vaccination. We found an 11- to 123-fold increase in antibodies binding to SARS-CoV and MERS-CoV as well as a 2- to 4-fold difference in antibodies binding to seasonal hCoVs in COVID-19 convalescent sera compared to pre-pandemic healthy donors, with the S2 subdomain of the S protein being the main target for cross-reactivity. In addition, we detected cross-reactive antibodies to all hCoV S proteins after SARS-CoV-2 vaccination in macaques and humans, with higher responses for hCoV more closely related to SARS-CoV-2. These findings support the feasibility of and provide guidance for development of a pan-coronavirus vaccine.
Abstract Comprehensive insight into the human gut microbiota and the interaction with their environment in communities with a high background of antibiotic use and antibiotic resistance genes is currently largely lacking. In a cohort (Vietnam), individuals within the same household, also individuals within their geographical cluster share more bacterial taxa than individuals from different households or geographical clusters. The microbial diversity among individuals who used antibiotics in the past four months was significantly lower than those who did not. Fecal microbiota of humans was more diverse than non-human samples, shared a small part of its amplicon sequence variants (ASVs) with feces from animals (7.4%), water (2.2%) and food (3.1%). Sharing of ASVs between humans and companion animals was not associated with household. There is a correlation between an Enterobacteriaceae ASV and the presence of blactx-m-2 in feces from humans and animals, hinting towards an exchange of antimicrobial resistant strains between reservoirs.
The emergence of SARS-CoV-2 variants that are more resistant to antibody-mediated neutralization pose a new hurdle in combating the COVID-19 pandemic. Although vaccines based on the original Wuhan sequence have been shown to be effective at preventing COVID-19, their efficacy is likely to be decreased against more neutralization-resistant variants-of-concern (VOC), in particular, the Beta variant originating in South Africa. We assessed, in mice, rabbits, and non-human primates, whether a third vaccination with experimental Wuhan-based Spike vaccines could alleviate this problem. Our data show that a third immunization improves neutralizing antibody titers against the variants-of-concern, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). After three vaccinations, the level of neutralization against Beta was similar to the level of neutralization against the original strain after two vaccinations, suggesting that simply providing a third immunization could nullify the reduced activity of current vaccines against VOC.
The urgent need for, but limited availability of, SARS-CoV-2 vaccines worldwide has led to widespread consideration of dose-sparing strategies. Here, we evaluate the SARS-CoV-2-specific antibody responses following BNT162b2 vaccination in 150 previously SARS-CoV-2-infected individuals from a population-based cohort. One week after first vaccine dose, spike protein antibody levels are 27-fold higher and neutralizing antibody titers 12-fold higher, exceeding titers of fully vaccinated SARS-CoV-2-naive controls, with minimal additional boosting after the second dose. Neutralizing antibody titers against four variants of concern increase after vaccination; however, overall neutralization breadth does not improve. Pre-vaccination neutralizing antibody titers and time since infection have the largest positive effect on titers following vaccination. COVID-19 severity and the presence of comorbidities have no discernible impact on vaccine response. In conclusion, a single dose of BNT162b2 vaccine up to 15 months after SARS-CoV-2 infection offers higher neutralizing antibody titers than 2 vaccine doses in SARS-CoV-2-naive individuals.
Antibodies against seasonal human coronaviruses (HCoVs) are known to cross-react with SARS-CoV-2, but data on cross-protective effects of prior HCoV infections are conflicting. In a prospective cohort of healthcare workers (HCW), we studied the association between seasonal HCoV (OC43, HKU1, 229E and NL63) nucleocapsid protein IgG and SARS-CoV-2 infection during the first pandemic wave in the Netherlands (March 2020- June 2020), by 4-weekly serum sampling. HCW with HCoV-OC43 antibody levels in the highest quartile, were less likely to become SARS-CoV-2 seropositive when compared to those with lower levels (6/32, 18.8%, vs 42/97, 43.4%, respectively: p=0.019; HR 0.37, 95% CI 0.16-0.88). We found no significant association with HCoV-OC43 spike protein IgG, or with antibodies against other HCoVs. Our results indicate that a recent infection with HCoV-OC43 has a protective effect against SARS-CoV-2 infection; this supports and informs efforts to develop pancoronavirus vaccines.Funding Information: This work was supported by the Netherlands Organization for Scientific Research (NWO) ZonMw (S3 study, grant agreement no. 10430022010023 to M.K.B.) and the Corona Research Fund Amsterdam UMC.Conflict of Interests: The authors declare no competing interests.Ethical Approval: The study was approved by the medical ethics review committee of both hospitals, and written informed consent was obtained from each participant.
To optimise the use of available SARS-CoV-2 vaccines, some advocate delaying second vaccination for individuals infected within six months. We studied whether post-vaccination immune response is equally potent in individuals infected over six months prior to vaccination.
Abstract Current SARS-CoV-2 vaccines are losing efficacy against emerging variants and may not protect against future novel coronavirus outbreaks, emphasizing the need for more broadly protective vaccines. To inform the development of a pan-coronavirus vaccine, we investigated the presence and specificity of cross-reactive antibodies against the spike (S) proteins of human coronaviruses (hCoV) after SARS-CoV-2 infection and vaccination. We found an 11 to 123-fold increase in antibodies binding to SARS-CoV and MERS-CoV as well as a 2 to 4-fold difference in antibodies binding to seasonal hCoVs in COVID-19 convalescent sera compared to pre-pandemic healthy donors, with the S2 subdomain of the S protein being the main target for cross-reactivity. In addition, we detected cross-reactive antibodies to all hCoV S proteins after SARS-CoV-2 S protein immunization in macaques, with higher responses for hCoV more closely related to SARS-CoV-2. These findings support the feasibility of and provide guidance for development of a pan-coronavirus vaccine.
