PIKA Provides an Adjuvant Effect to Induce Strong Mucosal and Systemic Humoral Immunity Against SARS-CoV

Severe Acute Respiratory Syndrome （SARS） is a deadly infectious disease caused by SARS Coronavirus （SARS-CoV）. Inactivated SARS-CoV has been explored as a vaccine against SARS-CoV. However, safe and potent adjuvants, especially with more efficient and economical needle-free vaccination are alw needed more urgently in a pandemic. The development of a safe and effective mucosal adjuvant and vaccine ays for prevention of emergent infectious diseases such as SARS will be an important advancement. PIKA, a stabilized derivative of Poly （I：C）, was previously reported to be safe and potent as adjuvant in mouse models. In the present study, we demonstrated that the intraperitoneal and intranasal co-administration of inactivated SARS-CoV vaccine together with this improved Poly （I：C） derivative induced strong anti-SARS-CoV mucosal and systemic humoral immune responses with neutralizing activity against pseudotyped virus. Although intraperitoneal immunization of inactivated SARS-CoV vaccine alone could induce a certain level of neutralizing activity in serum as well as in mucosal sites, co-administration of inactivated SARS-CoV vaccine with PIKA as adjuvant could induce a much higher neutralizing activity. When intranasal immunization was used, PIKA was obligatorily for inducing neutralizing activity in serum as well as in mucosal sites and was correlated with both mucosal IgA and mucosal IgG response. Overall, PIKA could be a good mucosal adjuvant candidate for inactivated SARS-CoV vaccine for use in possible future pandemic.

Nasal vaccination of triple-RBD scaffold protein with flagellin elicits long-term protection against SARS-CoV-2 variants including JN.1

Developing a mucosal vaccine against SARS-CoV-2 is critical for combatting the epidemic.Here,we investigated long-term immune responses and protection against SARS-CoV-2 for the intranasal vaccination of a triple receptor-binding domain(RBD)scaffold protein(3R-NC)adjuvanted with a flagellin protein(KFD)(3R-NC+KFDi.n).In mice,the vaccination elicited RBD-specific broad-neutralizing antibody responses in both serum and mucosal sites sustained at high level over a year.This long-lasting humoral immunity was correlated with the presence of long-lived RBD-specific IgG-and IgA-producing plasma cells,alongside the Th17 and Tfh17-biased T-cell responses driven by the KFD adjuvant.Based upon these preclinical findings,an open labeled clinical trial was conducted in individuals who had been primed with the inactivated SARS-CoV-2(IAV)vaccine.With a favorable safety profile,the 3R-NC+KFDi.n boost elicited enduring broad-neutralizing IgG in plasma and IgA in salivary secretions.To meet the challenge of frequently emerged variants,we further designed an updated triple-RBD scaffold protein with mutated RBD combinations,which can induce adaptable antibody responses to neutralize the newly emerging variants,including JN.1.Our findings highlight the potential of the KFD-adjuvanted triple-RBD scaffold protein is a promising prototype for the development of a mucosal vaccine against SARS-CoV-2 infection.

The protective nasal boosting of a triple-RBD subunit vaccine against SARS-CoV-2 following inactivated virus vaccination

Dear Editor,Though COVID-19 vaccines have been developed and clinically deployed rapidly,new variants of concern(VOCs)are still emerging frequently and escalating around the world.More breakthrough infections occurred even vaccination rates are high.For possible ending of the pandemic,curbing infection and stopping transmission are priority.

A triple-RBD-based mucosal vaccine provides broad protectionagainst SARS-CoV-2 variants of concern

The rapid mutation and spread of SARS-CoV-2 variants urge the development of effective mucosal vaccines to provide broadspectrum protection against the initial infection and thereby curb the transmission potential.Here,we designed a chimeric tripleRBD immunogen,3Ro-NC,harboring one Delta RBD and two Omicron RBDs within a novel protein scaffold.3Ro-NC elicits potent and broad RBD-specific neutralizing immunity against SARS-CoV-2 variants of concern.Notably,intranasal immunization with 3RoNC plus the mucosal adjuvant KFD(3Ro-NC+KFDi.n)elicits coordinated mucosal IgA and higher neutralizing antibody specificity(closer antigenic distance)against the Omicron variant.In Omicron-challenged human ACE2 transgenic mice,3Ro-NC+KFDi.n immunization significantly reduces the tissue pathology in the lung and lowers the viral RNA copy numbers in both the lung(85.7-fold)and the nasal turbinate(13.6-fold).Nasal virologic control is highly correlated with RBD-specific secretory IgA antibodies.Our data show that 3Ro-NC plus KFD is a promising mucosal vaccine candidate for protection against SARS-CoV-2 Omicron infection,pathology and transmission potential.

Rapid isolation and immune profiling of SARS-CoV-2 specific memory B cell in convalescent COVID-19 patients via LIBRA-seq

B cell response plays a critical role against SARS-CoV-2 infection.However,little is known about the diversity and frequency of the paired SARS-CoV-2 antigen-specific BCR repertoire after SARS-CoV-2 infection.Here,we performed single-cell RNA sequencing and VDJ sequencing using the memory and plasma B cells isolated from five convalescent COVID-19 patients,and analyzed the spectrum and transcriptional heterogeneity of antibody immune responses.Via linking BCR to antigen specificity through sequencing(LIBRA-seq),we identified a distinct activated memory B cell subgroup(CD11c^(high) CD95^(high))had a higher proportion of SARS-CoV-2 antigen-labeled cells compared with memory B cells.Our results revealed the diversity of paired BCR repertoire and the non-stochastic pairing of SARS-CoV-2 antigen-specific immunoglobulin heavy and light chains after SARS-CoV-2 infection.The public antibody clonotypes were shared by distinct convalescent individuals.Moreover,several antibodies isolated by LIBRA-seq showed high binding affinity against SARS-CoV-2 receptor-binding domain(RBD)or nucleoprotein(NP)via ELISA assay.Two RBD-reactive antibodies C14646P3S and C2767P3S isolated by LIBRA-seq exhibited high neutralizing activities against both pseudotyped and authentic SARS-CoV-2 viruses in vitro.Our study provides fundamental insights into B cell response following SARS-CoV-2 infection at the single-cell level.

Identification of forensically important blow fly species （Diptera： Calliphoridae） in China by mitochondrial cytochrome oxidase I gene differentiation

Unambiguous and rapid sarcosaphagous insect species identification is an essential requirement for forensic investigations. Although some insect species are difficult to classify morphologically, they can be effectively identified using molecular methods based on similarity with abundant authenticated reference DNA sequences in local databases. However, local databases are still relatively incomplete in China because of the large land area with distinct regional conditions. In this study, 75 forensically important blow flies were collected from 23 locations in 16 Chinese provinces, and a 278-bp segment of the cytochrome oxidase subunit I gene of all specimens was successfully sequenced. Phylogenetic analysis of the sequenced segments showed that all Calliphorid specimens were properly assigned into nine species with relatively strong supporting values, thus indicating that the 278-bp cytochrome oxidase subunit one region is suitable for identification of Calliphorid species. The clear difference between intraspecific threshold and interspecific divergence confirmed the potential of this region for Calliphorid species identification, especially for distinguishing between morphologically similar species. Intraspecific geographic variations were observed in Lucilia sericata （Meigen, 1826） and Lucilia caesar （Linnaeus, 1758）.

Alterations in Phenotypes and Responses of T Cells Within 6 Months of Recovery from COVID-19: A Cohort Study

The COVID-19 pandemic,caused by the SARS-CoV-2 infection,is a global health crisis.While many patients have clinically recovered,little is known about long-term alterations in T cell responses of COVID-19 convalescents.In this study,T cell responses in peripheral blood mononuclear cells of a long-time COVID-19 clinically recovered(20–26 weeks)cohort(LCR)were measured via flow cytometry and ELISpot.The T cell responses of LCR were comparatively analyzed against an age and sex matched short-time clinically recovered(4–9 weeks)cohort(SCR)and a healthy donor cohort(HD).All volunteers were recruited from Wuhan Jinyintan Hospital,China.Phenotypic analysis showed that activation marker PD-1 expressing on CD4^(+)T cells of LCR was still significantly lower than that of HD.Functional analysis indicated that frequencies of Tc2,Th2 and Th17 in LCR were comparable to those of HD,but Tc17 was higher than that of HD.In LCR,compared to the HD,there were fewer IFN-c producing T cells but more IL-2 secreting T cells.In addition,the circulating Tfh cells in LCR were still slightly lower compared to HD,though the subsets composition had recovered.Remarkably,SARS-CoV-2 specific T cell responses in LCR were comparable to that of SCR.Collectively,T cell responses experienced long-term alterations in phenotype and functional potential of LCR cohort.However,after clinical recovery,SARS-CoV-2 specific T cell responses could be sustained at least for six months,which may be helpful in resisting re-infection。

Broad phenotypic alterations and potential dysfunction of lymphocytes in individuals clinically recovered from COVID-19

Although millions of patients have clinically recovered from COVID-19,little is known about the immune status of lymphocytes in these individuals.In this study,the peripheral blood mononuclear cells of a clinically recovered(CR)cohort were comparatively analyzed with those of an age-and sex-matched healthy donor cohort.We found that CD8^(+)T cells in the CR cohort had higher numbers of effector T cells and effector memory T cells but lower Tc1(IFN-γ^(+)),Tc2(IL-4^(+)),and Tc17(IL-17A^(+))cell frequencies.The CD4^(+)T cells of the CR cohort were decreased in frequency,especially the central memory T cell subset.Moreover,CD4^(+)T cells in the CR cohort showed lower programmed cell death protein 1(PD-1)expression and had lower frequencies of Th1(IFN-γ^(+)),Th2(IL-4^(+)),Th17(IL-17A^(+)),and circulating follicular helper T(CXCR5^(+)PD-1^(+))cells.Accordingly,the proportion of isotype-switched memory B cells(IgM−CD20^(hi))among B cells in the CR cohort showed a significantly lower proportion,although the level of the activation marker CD71 was elevated.For CD3−HLA-DR−lymphocytes in the CR cohort,in addition to lower levels of IFN-γ,granzyme B and T-bet,the correlation between T-bet and IFN-γ was not observed.Additionally,by taking into account the number of days after discharge,all the phenotypes associated with reduced function did not show a tendency toward recovery within 4-11 weeks.The remarkable phenotypic alterations in lymphocytes in the CR cohort suggest that severe acute respiratory syndrome coronavirus 2 infection profoundly affects lymphocytes and potentially results in dysfunction even after clinical recovery.