ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6-specific nanobody restricts M. tuberculosis growth in macrophages.

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH-dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

Humoral Immunity Elicited by the XBB.1.5 Monovalent COVID-19 Vaccine.

As novel SARS-CoV-2 variants continue to emerge, the updated XBB.1.5 monovalent vaccines remain to be evaluated in terms of immunogenicity against live clinical isolates. We report boosting of IgG(2.1X), IgA(1.5X), and total IgG/A/M(1.7X) antibodies targeting the spike receptor-binding domain and neutralizing titers against WA1(2.2X), XBB.1.5(7.4X), EG.5.1(10.5X), and JN.1(4.7X) variants.

Evaluating Humoral Immunity Elicited by XBB.1.5 Monovalent COVID-19 Vaccine.

Because novel SARS-CoV-2 variants continue to emerge, immunogenicity of XBB.1.5 monovalent vaccines against live clinical isolates needs to be evaluated. We report boosting of IgG (2.1×), IgA (1.5×), and total IgG/A/M (1.7×) targeting the spike receptor-binding domain and neutralizing titers against WA1 (2.2×), XBB.1.5 (7.4×), EG.5.1 (10.5×), and JN.1 (4.7×) variants.

Diverging Maternal and Cord Antibody Functions From SARS-CoV-2 Infection and Vaccination in Pregnancy.

Maternal immunity impacts the infant, but how is unclear. To understand the implications of the immune exposures of vaccination and infection in pregnancy for neonatal immunity, we evaluated antibody functions in paired peripheral maternal and cord blood. We compared those who in pregnancy received mRNA coronavirus disease 2019 (COVID-19) vaccine, were infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the combination. We found that vaccination enriched a subset of neutralizing activities and Fc effector functions that was driven by IgG1 and was minimally impacted by antibody glycosylation in maternal blood. In paired cord blood, maternal vaccination also enhanced IgG1. However, Fc effector functions compared to neutralizing activities were preferentially transferred. Moreover, changes in IgG posttranslational glycosylation contributed more to cord than peripheral maternal blood antibody functional potency. These differences were enhanced with the combination of vaccination and infection as compared to either alone. Thus, Fc effector functions and antibody glycosylation highlight underexplored maternal opportunities to safeguard newborns.

ESAT-6 undergoes self-association at phagosomal pH and an ESAT-6 specific nanobody restricts M. tuberculosis growth in macrophages.

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

Diverging maternal and infant cord antibody functions from SARS-CoV-2 infection and vaccination in pregnancy.

Immunization in pregnancy is a critical tool that can be leveraged to protect the infant with an immature immune system but how vaccine-induced antibodies transfer to the placenta and protect the maternal-fetal dyad remains unclear. Here, we compare matched maternal-infant cord blood from individuals who in pregnancy received mRNA COVID-19 vaccine, were infected by SARS-CoV-2, or had the combination of these two immune exposures. We find that some but not all antibody neutralizing activities and Fc effector functions are enriched with vaccination compared to infection. Preferential transport to the fetus of Fc functions and not neutralization is observed. Immunization compared to infection enriches IgG1-mediated antibody functions with changes in antibody post-translational sialylation and fucosylation that impact fetal more than maternal antibody functional potency. Thus, vaccine enhanced antibody functional magnitude, potency and breadth in the fetus are driven more by antibody glycosylation and Fc effector functions compared to maternal responses, highlighting prenatal opportunities to safeguard newborns as SARS-CoV-2 becomes endemic.

The time between vaccination and infection impacts immunity against SARS-CoV-2 variants.

As the COVID-19 pandemic continues, long-term immunity against SARS-CoV-2 will be globally important. Official weekly cases have not dropped below 2 million since September of 2020, and continued emergence of novel variants have created a moving target for our immune systems and public health alike. The temporal aspects of COVID-19 immunity, particularly from repeated vaccination and infection, are less well understood than short-term vaccine efficacy. In this study, we explore the impact of combined vaccination and infection, also known as hybrid immunity, and the timing thereof on the quality and quantity of antibodies produced by a cohort of 96 health care workers. We find robust neutralizing antibody responses among those with hybrid immunity against all variants, including Omicron BA.2, and we further found significantly improved neutralizing titers with longer vaccine-infection intervals up to 400 days. These results indicate that anti-SARS-CoV-2 antibody responses undergo continual maturation following primary exposure by either vaccination or infection for at least 400 days after last antigen exposure. We show that neutralizing antibody responses improved upon secondary boosting with greater impact seen after extended intervals. Our findings may also extend to booster vaccine doses, a critical consideration in future vaccine campaign strategies.

An extended interval between vaccination and infection enhances hybrid immunity against SARS-CoV-2 variants.

As the COVID-19 pandemic continues, long-term immunity against SARS-CoV-2 will be important globally. Official weekly cases have not dropped below 2 million since September of 2020, and continued emergence of novel variants has created a moving target for our immune systems and public health alike. The temporal aspects of COVID-19 immunity, particularly from repeated vaccination and infection, are less well understood than short-term vaccine efficacy. In this study, we explored the effect of combined vaccination and infection, also known as hybrid immunity, and the timing thereof on the quality and quantity of antibodies elicited in a cohort of 96 health care workers. We found robust neutralizing antibody responses among those with hybrid immunity; these hybrid immune responses neutralized all variants, including BA.2. Neutralizing titers were significantly improved for those with longer vaccine-infection intervals of up to 400 days compared with those with shorter intervals. These results indicate that anti-SARS-CoV-2 antibody responses undergo continual maturation following primary exposure by either vaccination or infection for at least 400 days after last antigen exposure. We show that neutralizing antibody responses improved upon secondary boosting, with greater potency seen after extended intervals. Our findings may also extend to booster vaccine doses, a critical consideration in future vaccine campaign strategies.

Omicron neutralizing antibody response following booster vaccination compared with breakthrough infection.

BACKGROUND: The spread of the vaccine-resistant Omicron severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants threatens unvaccinated and fully vaccinated individuals, and accelerated booster vaccination campaigns are underway to mitigate the ongoing wave of Omicron cases. The immunity provided by standard vaccine regimens, boosted regimens, and immune responses elicited by vaccination plus natural infection remain incompletely understood. The magnitude, quality, and durability of serological responses, and the likelihood of protection against future SARS-CoV-2 variants following these modes of exposure, are poorly characterized but are critical to the future trajectory of the coronavirus disease 2019 (COVID-19) pandemic. METHODS: Ninety-nine individuals were semi-randomly selected from a larger vaccination cohort following vaccination and, in some cases, breakthrough infection. We analyzed spike receptor-binding domain-specific immunoglobulin G (IgG), IgA, and IgM by enzyme-linked immunosorbent assay, neutralizing antibody titers against live SARS-CoV-2 variants, and antibody-dependent cell-mediated phagocytosis. FINDINGS: In 99 vaccinated adults, compared with responses after two doses of an mRNA regimen, the immune responses 3 months after a third vaccine dose and 1 month after breakthrough infection due to prior variants show dramatic increases in magnitude, potency, and breadth, including increased antibody-dependent cellular phagocytosis and robust neutralization of the currently circulating Omicron BA.2 variant. CONCLUSIONS: Boosters and natural infection substantially boost immune responses. As the number of Omicron sub-variant cases rise and as global vaccination and booster campaigns continue, an increasing proportion of the world's population will acquire potent immune responses that may be protective against future SARS-CoV-2 variants. FUNDING: This work was funded by the M. J. Murdock Charitable Trust, the OHSU Foundation, the NIH (T32HL083808), and OHSU Innovative IDEA.

BNT162b2-induced neutralizing and non-neutralizing antibody functions against SARS-CoV-2 diminish with age.

Each severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant renews concerns about decreased vaccine neutralization weakening efficacy. However, while prevention of infection varies, protection from disease remains and implicates immunity beyond neutralization in vaccine efficacy. Polyclonal antibodies function through Fab domains that neutralize virus and Fc domains that induce non-neutralizing responses via engagement of Fc receptors on immune cells. To understand how vaccines promote protection, we leverage sera from 51 SARS-CoV-2 uninfected individuals after two doses of the BNT162b2 mRNA vaccine. We show that neutralizing activities against clinical isolates of wild-type and five SARS-CoV-2 variants, including Omicron BA.2, link to FcγRIIIa/CD16 non-neutralizing effector functions. This is associated with post-translational afucosylation and sialylation of vaccine-specific antibodies. Further, polyfunctional neutralizing and non-neutralizing breadth, magnitude, and coordination diminish with age. Thus, studying Fc functions in addition to Fab-mediated neutralization provides greater insight into vaccine efficacy for vulnerable populations, such as the elderly, against SARS-CoV-2 and novel variants.

BNT162b2 induced neutralizing and non-neutralizing antibody functions against SARSCoV-2 diminish with age.

Each novel SARS-CoV-2 variant renews concerns about decreased vaccine efficacy caused by evasion of vaccine induced neutralizing antibodies. However, accumulating epidemiological data show that while vaccine prevention of infection varies, protection from severe disease and death remains high. Thus, immune responses beyond neutralization could contribute to vaccine efficacy. Polyclonal antibodies function through their Fab domains that neutralize virus directly, and Fc domains that induce non-neutralizing host responses via engagement of Fc receptors on immune cells. To understand how vaccine induced neutralizing and non-neutralizing activities synergize to promote protection, we leverage sera from 51 SARS-CoV-2 uninfected health-care workers after two doses of the BNT162b2 mRNA vaccine. We show that BNT162b2 elicits antibodies that neutralize clinical isolates of wildtype and five variants of SARS-CoV-2, including Omicron BA.2, and, critically, induce Fc effector functions. FcγRIIIa/CD16 activity is linked to neutralizing activity and associated with post-translational afucosylation and sialylation of vaccine specific antibodies. Further, neutralizing and non-neutralizing functions diminish with age, with limited polyfunctional breadth, magnitude and coordination observed in those ≥65 years old compared to <65. Thus, studying Fc functions in addition to Fab mediated neutralization provides greater insight into vaccine efficacy for vulnerable populations such as the elderly against SARS-CoV-2 and novel variants.

A Single Dose of ChAdOx1 nCoV-19 Vaccine Elicits High Antibody Responses in Individuals with Prior SARS-CoV-2 Infection Comparable to That of Two-Dose-Vaccinated, SARS-CoV-2-Infection-Naïve Individuals: A Longitudinal Study in Ethiopian Health Workers.

Single-dose COVID-19 vaccines, mostly mRNA-based vaccines, are shown to induce robust antibody responses in individuals who were previously infected with SARS-CoV-2, suggesting the sufficiency of a single dose for those individuals in countries with limited vaccine supply. However, these important data are limited to developed nations. We conducted a prospective longitudinal study among Ethiopian healthcare workers who received a ChAdOx1 nCoV-19 vaccine. We compared the geometric mean titers (GMTs) of the SARS-CoV-2 receptor-binding domain (RBD)-specific IgG antibodies in 39 SARS-CoV-2 naïve participants and 24 participants previously infected with SARS-CoV-2 (P.I.), who received two doses of ChAdOx1 nCoV-19 vaccine across the two post-vaccination time points (at 8 to 12 weeks post single dose and two dose vaccinations). We noted that the GMT (1632.16) in naïve participants at 8-12 weeks post first dose were comparable to the GMT (1674.94) observed in P.I. participants prior to vaccination. Interestingly, P.I. participants had significantly higher antibody titers compared to naïve participants, after both the first (GMT, 4913.50 vs. 1632.16) and second doses (GMT, 9804.60 vs. 6607.30). Taken together, our findings show that a single ChAdOx1 nCoV-19 dose in previously SARS-CoV-2 infected individuals elicits similar, if not higher, antibody responses to those of two-dose-vaccinated naïve individuals.

A global lipid map reveals host dependency factors conserved across SARS-CoV-2 variants.

A comprehensive understanding of host dependency factors for SARS-CoV-2 remains elusive. Here, we map alterations in host lipids following SARS-CoV-2 infection using nontargeted lipidomics. We find that SARS-CoV-2 rewires host lipid metabolism, significantly altering hundreds of lipid species to effectively establish infection. We correlate these changes with viral protein activity by transfecting human cells with each viral protein and performing lipidomics. We find that lipid droplet plasticity is a key feature of infection and that viral propagation can be blocked by small-molecule glycerolipid biosynthesis inhibitors. We find that this inhibition was effective against the main variants of concern (alpha, beta, gamma, and delta), indicating that glycerolipid biosynthesis is a conserved host dependency factor that supports this evolving virus.

Omicron neutralizing antibody response following booster vaccination compared with breakthrough infection.

The rapid spread of the vaccine-resistant Omicron variant of SARS-CoV-2 presents a renewed threat to both unvaccinated and fully vaccinated individuals, and accelerated booster vaccination campaigns are underway to mitigate the ongoing wave of Omicron cases. The degree of immunity provided by standard vaccine regimens, boosted regimens, and immune responses elicited by the combination of vaccination and natural infection remain incompletely understood. The relative magnitude, quality and durability of serological responses, and the likelihood of neutralizing protection against future SARS-CoV-2 variants following these modes of exposure are unknown but are critical to the future trajectory of the COVID-19 pandemic. In this study of 99 vaccinated adults, we find that compared with responses after two doses of an mRNA regimen, the immune responses three months after a third vaccine dose and one month after breakthrough infection due to prior variants show dramatic increases in magnitude, potency, and breadth, including increased antibody dependent cellular phagocytosis and robust neutralization of the recently circulating Omicron variant. These results suggest that as the number of Omicron cases rise and as global vaccination and booster campaigns continue, an increasing proportion of the world’s population will acquire potent immune responses that may be protective against future SARS-CoV-2 variants.

High seroprevalence of anti-SARS-CoV-2 antibodies among Ethiopian healthcare workers.

BACKGROUND: COVID-19 pandemic has a devastating impact on the economies and health care system of sub-Saharan Africa. Healthcare workers (HWs), the main actors of the health system, are at higher risk because of their occupation. Serology-based estimates of SARS-CoV-2 infection among HWs represent a measure of HWs' exposure to the virus and could be used as a guide to the prevalence of SARS-CoV-2 in the community and valuable in combating COVID-19. This information is currently lacking in Ethiopia and other African countries. This study aimed to develop an in-house antibody testing assay, assess the prevalence of SARS-CoV-2 antibodies among Ethiopian high-risk frontline HWs. METHODS: We developed and validated an in-house Enzyme-Linked Immunosorbent Assay (ELISA) for specific detection of anti-SARS-CoV-2 receptor binding domain immunoglobin G (IgG) antibodies. We then used this assay to assess the seroprevalence among HWs in five public hospitals located in different geographic regions of Ethiopia. From consenting HWs, blood samples were collected between December 2020 and February 2021, the period between the two peaks of COVID-19 in Ethiopia. Socio-demographic and clinical data were collected using questionnaire-based interviews. Descriptive statistics and bivariate and multivariate logistic regression were used to determine the overall and post-stratified seroprevalence and the association between seropositivity and potential risk factors. RESULTS: Our successfully developed in-house assay sensitivity was 100% in serum samples collected 2- weeks after the first onset of symptoms whereas its specificity in pre-COVID-19 pandemic sera was 97.7%. Using this assay, we analyzed a total of 1997 sera collected from HWs. Of 1997 HWs who provided a blood sample, and demographic and clinical data, 51.7% were females, 74.0% had no symptoms compatible with COVID-19, and 29.0% had a history of contact with suspected or confirmed patients with SARS-CoV-2 infection. The overall seroprevalence was 39.6%. The lowest (24.5%) and the highest (48.0%) seroprevalence rates were found in Hiwot Fana Specialized Hospital in Harar and ALERT Hospital in Addis Ababa, respectively. Of the 821 seropositive HWs, 224(27.3%) of them had a history of symptoms consistent with COVID-19 while 436 (> 53%) of them had no contact with COVID-19 cases as well as no history of COVID-19 like symptoms. A history of close contact with suspected/confirmed COVID-19 cases is associated with seropositivity (Adjusted Odds Ratio (AOR) = 1.4, 95% CI 1.1-1.8; p = 0.015). CONCLUSION: High SARS-CoV-2 seroprevalence levels were observed in the five Ethiopian hospitals. These findings highlight the significant burden of asymptomatic infection in Ethiopia and may reflect the scale of transmission in the general population.

A global lipid map reveals host dependency factors conserved across SARS-CoV-2 variants.

A comprehensive understanding of host dependency factors for SARS-CoV-2 remains elusive. We mapped alterations in host lipids following SARS-CoV-2 infection using nontargeted lipidomics. We found that SARS-CoV-2 rewires host lipid metabolism, altering 409 lipid species up to 64-fold relative to controls. We correlated these changes with viral protein activity by transfecting human cells with each viral protein and performing lipidomics. We found that lipid droplet plasticity is a key feature of infection and that viral propagation can be blocked by small-molecule glycerolipid biosynthesis inhibitors. We found that this inhibition was effective against the main variants of concern (alpha, beta, gamma, and delta), indicating that glycerolipid biosynthesis is a conserved host dependency factor that supports this evolving virus.

A potent alpaca-derived nanobody that neutralizes SARS-CoV-2 variants.

The spike glycoprotein of SARS-CoV-2 engages with human ACE 2 to facilitate infection. Here, we describe an alpaca-derived heavy chain antibody fragment (VHH), saRBD-1, that disrupts this interaction by competitively binding to the spike protein receptor-binding domain. We further generated an engineered bivalent nanobody construct engineered by a flexible linker and a dimeric Fc conjugated nanobody construct. Both multivalent nanobodies blocked infection at picomolar concentrations and demonstrated no loss of potency against emerging variants of concern including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Epsilon (B.1.427/429), and Delta (B.1.617.2). saRBD-1 tolerates elevated temperature, freeze-drying, and nebulization, making it an excellent candidate for further development into a therapeutic approach for COVID-19.

Cannabinoids Block Cellular Entry of SARS-CoV-2 and the Emerging Variants.

As a complement to vaccines, small-molecule therapeutic agents are needed to treat or prevent infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, which cause COVID-19. Affinity selection-mass spectrometry was used for the discovery of botanical ligands to the SARS-CoV-2 spike protein. Cannabinoid acids from hemp (Cannabis sativa) were found to be allosteric as well as orthosteric ligands with micromolar affinity for the spike protein. In follow-up virus neutralization assays, cannabigerolic acid and cannabidiolic acid prevented infection of human epithelial cells by a pseudovirus expressing the SARS-CoV-2 spike protein and prevented entry of live SARS-CoV-2 into cells. Importantly, cannabigerolic acid and cannabidiolic acid were equally effective against the SARS-CoV-2 alpha variant B.1.1.7 and the beta variant B.1.351. Orally bioavailable and with a long history of safe human use, these cannabinoids, isolated or in hemp extracts, have the potential to prevent as well as treat infection by SARS-CoV-2.

Vaccination before or after SARS-CoV-2 infection leads to robust humoral response and antibodies that effectively neutralize variants.

Current coronavirus disease 2019 (COVID-19) vaccines effectively reduce overall morbidity and mortality and are vitally important to controlling the pandemic. Individuals who previously recovered from COVID-19 have enhanced immune responses after vaccination (hybrid immunity) compared with their naïve-vaccinated peers; however, the effects of post-vaccination breakthrough infections on humoral immune response remain to be determined. Here, we measure neutralizing antibody responses from 104 vaccinated individuals, including those with breakthrough infections, hybrid immunity, and no infection history. We find that human immune sera after breakthrough infection and vaccination after natural infection broadly neutralize SARS-CoV-2 (severe acute respiratory coronavirus 2) variants to a similar degree. Although age negatively correlates with antibody response after vaccination alone, no correlation with age was found in breakthrough or hybrid immune groups. Together, our data suggest that the additional antigen exposure from natural infection substantially boosts the quantity, quality, and breadth of humoral immune response regardless of whether it occurs before or after vaccination.

A single dose ChAdOx1 nCoV-19 vaccine elicits high antibody responses in individuals with prior SARS-CoV-2 infection comparable to that of double dose vaccinated SARS-CoV-2 infection naïve individuals.

Background A single dose COVID-19 vaccines, mostly mRNA-based vaccines, are shown to induce robust antibody responses in individuals who were previously infected with SARS-CoV-2, suggesting the sufficiency of a single dose to those individuals. However, these important data are limited to developed nations and lacking in resource-limited countries, like Ethiopia. Methods We compared receptor-binding domain (RBD)-specific IgG antibodies in 40 SARS-CoV-2 naïve participants and 25 participants previously infected with SARS-CoV-2, who received two doses of ChAdOx1 nCoV-19 vaccine. We measured the antibody response in post-vaccination blood samples from both groups of participants collected at four different post-vaccination time points: 8- and 12-weeks after each dose of the vaccine administration using an in-house developed ELISA. Results We observed a high level of anti-RBD IgG antibodies titers 8-weeks after a single dose administration (16/27; 59.3%) among naïve participants, albeit dropped significantly (p<0.05) two months later, suggesting the protective immunity elicited by the first dose ChAdOx1 nCoV-19 vaccine will likely last for a minimum of three months. However, as expected, a significant (p<0.001) increase in the level of anti-RBD IgG antibodies titers was observed after the second dose administration in all naïve participants. By contrast, the ChAdOx1 nCoV-19 vaccine-induced anti-RBD IgG antibody titers produced by the P.I participants at 8- to 12-weeks post-single dose vaccination were found to be similar to the antibody titers seen after a two-dose vaccination course among infection- naïve participants and showed no significant (p>0.05) increment following the second dose administration. Conclusion Taken together, our findings show that a single ChAdOx1 nCoV-19 dose in previously SARS-CoV-2 infected individuals elicits similar antibody responses to that of double dose vaccinated naïve individuals. Age and sex were not associated with the level of vaccine-elicited immune responses in both individuals with and without prior SARS-CoV-2 infection. Further studies are required to assess the need for a booster dose to extend the duration and amplitude of the specific protective immune response in Ethiopia settings, especially following the Omicron pandemic.