SARS-CoV-2-specific nasal IgA wanes 9 months after hospitalisation with COVID-19 and is not induced by subsequent vaccination

BACKGROUND: Most studies of immunity to SARS-CoV-2 focus on circulating antibody, giving limited insights into mucosal defences that prevent viral replication and onward transmission. We studied nasal and plasma antibody responses one year after hospitalisation for COVID-19, including a period when SARS-CoV-2 vaccination was introduced. METHODS: In this follow up study, plasma and nasosorption samples were prospectively collected from 446 adults hospitalised for COVID-19 between February 2020 and March 2021 via the ISARIC4C and PHOSP-COVID consortia. IgA and IgG responses to NP and S of ancestral SARS-CoV-2, Delta and Omicron (BA.1) variants were measured by electrochemiluminescence and compared with plasma neutralisation data. FINDINGS: Strong and consistent nasal anti-NP and anti-S IgA responses were demonstrated, which remained elevated for nine months (p < 0.0001). Nasal and plasma anti-S IgG remained elevated for at least 12 months (p < 0.0001) with plasma neutralising titres that were raised against all variants compared to controls (p < 0.0001). Of 323 with complete data, 307 were vaccinated between 6 and 12 months; coinciding with rises in nasal and plasma IgA and IgG anti-S titres for all SARS-CoV-2 variants, although the change in nasal IgA was minimal (1.46-fold change after 10 months, p = 0.011) and the median remained below the positive threshold determined by pre-pandemic controls. Samples 12 months after admission showed no association between nasal IgA and plasma IgG anti-S responses (R = 0.05, p = 0.18), indicating that nasal IgA responses are distinct from those in plasma and minimally boosted by vaccination. INTERPRETATION: The decline in nasal IgA responses 9 months after infection and minimal impact of subsequent vaccination may explain the lack of long-lasting nasal defence against reinfection and the limited effects of vaccination on transmission. These findings highlight the need to develop vaccines that enhance nasal immunity. FUNDING: This study has been supported by ISARIC4C and PHOSP-COVID consortia. ISARIC4C is supported by grants from the National Institute for Health and Care Research and the Medical Research Council. Liverpool Experimental Cancer Medicine Centre provided infrastructure support for this research. The PHOSP-COVD study is jointly funded by UK Research and Innovation and National Institute of Health and Care Research. The funders were not involved in the study design, interpretation of data or the writing of this manuscript

Large-scale phenotyping of patients with long COVID post-hospitalization reveals mechanistic subtypes of disease

One in ten severe acute respiratory syndrome coronavirus 2 infections result in prolonged symptoms termed long coronavirus disease (COVID), yet disease phenotypes and mechanisms are poorly understood1. Here we profiled 368 plasma proteins in 657 participants ≥3 months following hospitalization. Of these, 426 had at least one long COVID symptom and 233 had fully recovered. Elevated markers of myeloid inflammation and complement activation were associated with long COVID. IL-1R2, MATN2 and COLEC12 were associated with cardiorespiratory symptoms, fatigue and anxiety/depression; MATN2, CSF3 and C1QA were elevated in gastrointestinal symptoms and C1QA was elevated in cognitive impairment. Additional markers of alterations in nerve tissue repair (SPON-1 and NFASC) were elevated in those with cognitive impairment and SCG3, suggestive of brain–gut axis disturbance, was elevated in gastrointestinal symptoms. Severe acute respiratory syndrome coronavirus 2-specific immunoglobulin G (IgG) was persistently elevated in some individuals with long COVID, but virus was not detected in sputum. Analysis of inflammatory markers in nasal fluids showed no association with symptoms. Our study aimed to understand inflammatory processes that underlie long COVID and was not designed for biomarker discovery. Our findings suggest that specific inflammatory pathways related to tissue damage are implicated in subtypes of long COVID, which might be targeted in future therapeutic trials

LT-IIb(T13I), a non-toxic type II heat-labile enterotoxin, augments the capacity of a ricin toxin subunit vaccine to evoke neutralizing antibodies and protective immunity.

Currently, there is a shortage of adjuvants that can be employed with protein subunit vaccines to enhance protection against biological threats. LT-IIb(T13I) is an engineered nontoxic derivative of LT-IIb, a member of the type II subfamily of heat labile enterotoxins expressed by Escherichia coli, that possesses potent mucosal adjuvant properties. In this study we evaluated the capacity of LT-IIb(T13I) to augment the potency of RiVax, a recombinant ricin toxin A subunit vaccine, when co-administered to mice via the intradermal (i.d.) and intranasal (i.n.) routes. We report that co-administration of RiVax with LT-IIb(T13I) by the i.d. route enhanced the levels of RiVax-specific serum IgG antibodies (Ab) and elevated the ratio of ricin-neutralizing to non-neutralizing Ab, as compared to RiVax alone. Protection against a lethal ricin challenge was also augmented by LT-IIb(T13I). While local inflammatory responses elicited by LT-IIb(T13I) were comparable to those elicited by aluminum salts (Imject®), LT-IIb(T13I) was more effective than aluminum salts at augmenting production of RiVax-specific serum IgG. Finally, i.n. administration of RiVax with LT-IIb(T13I) also increased levels of RiVax-specific serum and mucosal Ab and enhanced protection against ricin challenge. Collectively, these data highlight the potential of LT-IIb(T13I) as an effective next-generation i.d., or possibly i.n. adjuvant for enhancing the immunogenicity of subunit vaccines for biodefense

LT-IIb and LT-IIb(T13I) enhance the immune response to RiVax when co-administered by the i.d. route.

<p>Mice were immunized intradermally with 5.0 µg of RiVax alone or with 1.0 µg of LT-IIb or LT-IIb(T13I) on days 0, 10, and 20. (A) Level of Ag-specific IgG Abs in sera from immunized mice on days 7, 17, and 27. (B) Level of RiVax-specific IgG and IgA Abs obtained on day 27 in BAL, salivary, and fecal samples from immunized mice. Data (n = 5) presented as the arithmetic mean with error bars denoting one S.E.M on a logarithmic scale (Y-axis). Key: *, p<0.05; **, p<0.01; ***, p<0.001 compared to RiVax alone. Data were compared using an unpaired Students t-test.</p

LT-IIb(T13I) is minimally inflammatory in comparison to LT-IIb.

<p>Mice were immunized by the i.d. route with 5.0 µg of RiVax, RiVax adsorbed to Imject®(alum), or RiVax combined with 1.0 µg of either LT-IIb or LT-IIb(T13I). (A) Gross morphologic photos and H&E stained micrographs of typical inflammation at the site of injection on day 7 post-immunization (n = 5). (B) Level of inflammatory induration at the injection site (n = 5). (C) Percent of CD45⁺ immune cells normalized to the total number of cells counted using Axiovision and ImageJ software from 15 random fluorescent micrographs per skin section (n = 3). Data are presented as the arithmetic mean with error bars denoting one S.E.M. Key: significance in comparison to LT-IIb(T13I) *, p<0.05; **, p<0.01; ***, p<0.001. Data in B were compared using an unpaired Student’s t-test; data in C were compared using ANOVA; #, p<0.05 - significance in comparison to LT-IIb; **, p<0.01 - significance in comparison to RiVax.</p

Administration of LT-IIb(T13I) by the i.d. route enhances protective immunity to ricin.

<p>Mice were immunized intradermally on days 0, 10, and 20 with 0.5 µg of RiVax in the presence or absence of 1.0 µg of LT-IIb(T13I) and challenged 14 days after the final immunization with 10 LD₅₀ of ricin. (A) Survival of immunized mice after i.p. challenge with ricin. Data were compared by the Logrank test. (B) Blood glucose levels of immunized mice during challenge with ricin. Data presented as the arithmetic mean with error bars denoting one S.E.M. (n = 5). Key: *, p<0.05; **, p<0.01 compared to RiVax; †<b>,</b> p<0.05 compared to 0 h. Data were compared using an unpaired Student’s t-test.</p

LT-IIb(T13I) is superior to alum (Imject®) at enhancing immune responses to RiVax when administered intradermally.

<p>Mice were immunized intradermally on days 0, 10, and 20 with 0.5 µg of RiVax, RiVax adsorbed to Imject®, or RiVax combined with 1.0 µg of either LT-IIb or LT-IIb(T13I). (A) Level of RiVax-specific serum IgG Ab from days 17, 27, 60, 90, and 120. (B) RiVax-specific IgG subclass analysis from the sera of immunized mice taken on day 90. Data are presented as the arithmetic mean with error bars denoting one S.E.M. (n = 5). Key: (A) *, p<0.05; **, p<0.01; ***, p<0.001 compared to RiVax; #, p<0.05 compared to RiVax+alum. (B) *, p<0.001 compared to IgG2a and IgG2b from the same group. Data were compared using ANOVA.</p

I.d. Immunization of RiVax with LT-IIb or LT-IIb(T13I) enhances ricin-neutralizing Ab production.

<p>Sera from immunized mice were assessed for the capacity to neutralize ricin in a Vero cell cytotoxicity assay. Ricin (10 ng/mL) was incubated with serum for 30 min and the mixture was applied in triplicate to Vero cells grown in 96-well microtiter plates for 2 h at 37°C. After washing, fresh media was applied and cell viability was assessed 48 h later. Data shown is representative for each group of immunized animals (n = 5) with the error bars denoting one S.E.M. of the average of three replicate wells of treated Vero cells.</p

Immunization with LT-IIb(T13I) by the i.n. route enhances RiVax-specific Ab production.

<p>Mice were immunized intranasally with 5.0 µg of RiVax alone or with 1.0 µg of LT-IIb or LT-IIb(T13I) on days 0, 10, and 20. (A) Level of RiVax-specific IgG Ab in sera from immunized mice on days 7, 17, and 27. (B) Level of RiVax-specific IgG and IgA Ab obtained on day 27 from lung lavage, saliva, and fecal samples from immunized mice. Data (n = 5) are presented as the arithmetic mean with error bars denoting one S.E.M on a logarithmic scale (Y-axis). Key: *, p<0.05; **, p<0.01; ***, p<0.001 compared to RiVax. Data were compared using an unpaired Student’s t-test.</p

I.n. immunization of RiVax with LT-IIb(T13I) enhances protective immunity to ricin.

<p>Mice were immunized intranasally on days 0, 10, 20, and 34 with 0.5 µg of RiVax in the presence or absence of 1.0 µg of LT-IIb or LT-IIb(T13I) and challenged 14 days after the final immunization with 10 LD₅₀ of ricin. (A) Survival of mice after i.p. challenge with ricin. Data were compared by the Logrank test. (B) Blood glucose levels of immunized mice during ricin challenge. Data are presented as the arithmetic mean with error bars denoting one S.E.M. (n = 4 or 5) Key: **, p<0.01 compared to RiVax; †, p<0.01 compared to 0 h. Data were compared using an unpaired Students t-test.</p