Limited Tryptic Digestion-Isotope Dilution Mass Spectrometry (LTD-IDMS): A Reagent-Free Analytical Assay To Quantify Hemagglutinin of A(H5N1) Vaccine Material.

Avian influenza viruses, such as A(H5N1) and A(H7N9), are primary public health concerns due to their pandemic potential. Influenza vaccines represent the most effective response to this threat especially with timely provision. The current pandemic response timelines require a substantial period for strain-specific reference antigen and sera preparation for use with single-radial immunodiffusion (SRID), the accepted vaccine potency assay. To address this time lag, the isotope dilution mass spectrometry (IDMS) method was developed to quantify the absolute hemagglutinin (HA, the main influenza antigen) amount in the vaccine without the need for purified, inactivated, and calibrated virus reference antigens. However, an additional challenge in determining potency is to differentiate between vaccine antigens in their most potent form from other less potent, stressed antigen forms. The limited trypsin digestion (LTD) method has been developed and does not require strain-specific full-length reference antigens or antibodies; instead, stressed HA is selectively degraded, leaving the more potent form to be measured. LTD, followed by precipitation and IDMS, allows for efficient differentiation between potent and significantly less potent HA for vaccine release and potency testing across the vaccine's shelf life. In this study, we tested the LTD-IDMS assay on A(H5N1) vaccine material that had been stressed by low pH, heat, and multiple freeze-thaw cycles. The results showed that the LTD-IDMS method effectively quantified the potent HA in A(H5N1) vaccine material with results comparable to SRID. As such, it shows great promise to complement and potentially replace SRID in a pandemic when strain-specific reagents may not be readily available.

Comparability of Titers of Antibodies against Seasonal Influenza Virus Strains as Determined by Hemagglutination Inhibition and Microneutralization Assays.

We compared titers of antibodies against A/H1N1, A/H3N2, and B influenza virus strains collected pre- and postvaccination using hemagglutination inhibition (HI) and microneutralization (MN) assays and data from two vaccine trials: study 1, performed with a cell-grown trivalent influenza vaccine (TIVc) using cell-grown target virus in both assays, and study 2, performed with an egg-grown adjuvanted quadrivalent influenza vaccine (aQIVe) using egg-grown target virus. The relationships between HI- and MN-derived log-transformed titers were examined using different statistical techniques. Deming regression analyses showed point estimates for slopes generally close to 1 across studies and strains. The slope of regression was closest to 1 for A/H3N2 strain when either cell- or egg-grown viral target virus was used. Bland-Altman plots indicated a very small percentage of results outside 2 and 3 standard deviations. The magnitudes and directions of differences between titers in the two assays varied by study and strain. Mean differences favored the MN assay for A/H1N1 and B strains in study 1, whereas the titers determined by HI were higher than those determined by MN against the A/H3N2 strain. In study 2, mean differences favored the MN assay for A/H3N2 and B strains. Overall, the directions and magnitudes of the mean differences were similar between the two vaccines. The concordance correlation coefficient values ranged from 0.74 (A/H1N1 strain, study 1) to 0.97 (A/H3N2 strain, study 1). The comparative analysis demonstrates an overall strong positive correlation between the HI and MN assays. These data support the use of the MN assay to quantify the immune response of influenza vaccines in clinical studies, particularly for the A/H3N2 strain.

Kidney Transplant Modifies the Architecture and Microenvironment of Basal Cell Carcinomas.

BACKGROUND/AIMS: Basal cell carcinoma (BCC) is a frequent type of nonmelanoma skin cancer, which shows a greater prevalence in kidney-transplanted (KT) patients than in the general population. The study of this tumor in KT patients may allow us to understand the influence of the tumor inflammatory microenvironment on cancer behavior, and to design new image analysis methods to determine prognosis and apply personalized medicine. The major hypothesis of the present work is that antirejection drugs, by modifying the B-cell/T-cell balance, induce measurable differences in tumoral cell microarchitecture and in the inflammatory microenvironment in KT patients compared to nontransplanted controls. METHODS: In this retrospective study in an Italian cohort including 15 KT patients and 15 control subjects from the general population who developed BCC, we analyzed tissue microarchitecture and inflammatory infiltrates of BCC using state-of-the-art nonlinear image analysis techniques such as fractal dimension and sample entropy of internuclear distances. RESULTS: KT patients showed a nonsignificant trend to a greater number of nuclei in the basal cell layer compared to non-KT controls and subtle changes in the intact skin compared to controls. Similarly, the number of mitoses per unit length was almost doubled in the patients with KT compared to controls. However, when the number of mitotic cells was normalized by the total number of cells in the basal layer (mitotic index), these differences were not significant, although a clear trend was still present. Finally, KT patients showed a nonsignificant trend to an increased -density of inflammatory cells close to the tumoral cell layer. When considering the intact skin, this difference was significant, with a 70% increase in the density of inflammatory cells. CONCLUSION: Data comparing the microarchitecture of BCC in normal subjects and KT patients are scanty, and the present study is the first to use nonlinear image analysis techniques to this aim. The observed differences underscore the relevance of T-cell suppression in cancer behavior. These data suggest that BCC develops in treated patients with specific biological characteristics which should be further analyzed in terms of therapeutic response.

Saliva for assessing creatinine, uric acid, and potassium in nephropathic patients.

BACKGROUND: Lab tests on saliva could be useful because of low invasivity. Previous reports indicated that creatinine, uric acid, and potassium are measurable in saliva. For these analytes the study investigated methodology of saliva tests and correlations between plasma and saliva levels. METHODS: The study enrolled 15 healthy volunteers for methodological analyses and 42 nephropathic patients for plasma-saliva correlations (35 non-dialysis and 7 dialysis). Saliva was collected by synthetic swap right after venipuncture for blood withdrawal. Blood and saliva, unless otherwise indicated, were collected early in the morning after overnight fast and lab tests were performed in fresh samples by automated biochemistry (standard). Methodological analyses included blind duplicates, different collection mouth sites, day-to-day variability, different collection times, and freezing-thawing effects. Analyses on plasma-saliva correlations included post-dialysis changes. RESULTS: For saliva lab tests of all analytes, blind duplicates, samples from different mouth sites or of different days were not significantly different but were significantly correlated (differences ≤14.4%; R ≥ 0.620, P ≤ 0.01). For all analytes, mid-morning saliva had lower levels than but correlated with standard saliva (differences ≥15.8%; R ≥ 0.728, P ≤ 0.01). Frozen-thawed saliva had lower levels than fresh saliva for uric acid only (- 17.2%, P < 0.001). Frozen-thawed saliva correlated with fresh saliva for all analytes (R ≥ 0.818, P ≤ 0.001). Saliva and plasma levels differed but correlated with plasma for creatinine (R = 0.874, P < 0.001), uric acid (R = 0.821, P < 0.001) and potassium (R = 0.767, P < 0.001). Post-dialysis changes in saliva paralleled post-dialysis changes in plasma. CONCLUSION: Saliva levels of creatinine, uric acid, and potassium are measurable and correlated with their plasma levels. Early morning fasting fresh saliva samples are advisable because later collection times or freezing lower the saliva levels of these analytes.

Salivary levels of phosphorus and urea as indices of their plasma levels in nephropathic patients.

BACKGROUND: Phosphorus and urea are measurable in saliva. Measurements of saliva phosphorus (S-Pho) and saliva urea (S-Urea) could be useful because of low invasivity. Data are limited to saliva tests methodology and to correlations between plasma and saliva compositions. S-Pho and S-Urea were investigated focusing on blind duplicates, differences between collection sites, differences between collection times, freezing-thawing effects, and plasma-saliva correlations. METHODS: Tests were performed using fresh saliva collected by synthetic swap early morning after overnight fast (standard). Methodology was investigated in fifteen healthy volunteers. Plasma-saliva correlations were investigated in thirty nephropathic outpatients. RESULTS: S-Pho and S-Urea in all measurements ranged above detection limits (0.3 mmol/L). In healthy volunteers, S-Pho and S-Urea were similar in duplicates (results for S-Pho and S-Urea: % difference between samples ≤ 4.85%; R between samples ≥ .976, P < .001), in samples from different mouth sites (≤4.24%; R ≥ .887, P < .001), and in samples of different days (≤5.61%; R ≥ .606, P < .01) but, compared to standard, were substantially lower in after-breakfast samples (-28.0% and -21.3%; R ≥ .786, P < .001) and slightly lower in frozen-thawed samples (-12.4% and -5.92%; R ≥ .742, P < .001). In nephropathic patients, S-Pho was higher than but correlated with plasma phosphorus (saliva/plasma ratio 4.80; R = .686, P < .001), whereas S-Urea and plasma urea were similar and correlated with each other (saliva/plasma ratio 0.96; R = .944, P < .001). Post-dialysis changes in S-Pho and S-Urea paralleled post-dialysis changes in plasma phosphorus and urea. CONCLUSION: S-Pho and S-Urea reflect plasma phosphorus and plasma urea. Early morning fasting fresh samples are advisable because collection time and freezing-thawing affect saliva tests.

A site of varicella-zoster virus vulnerability identified by structural studies of neutralizing antibodies bound to the glycoprotein complex gHgL.

Varicella-zoster virus (VZV), of the family Alphaherpesvirinae, causes varicella in children and young adults, potentially leading to herpes zoster later in life on reactivation from latency. The conserved herpesvirus glycoprotein gB and the heterodimer gHgL mediate virion envelope fusion with cell membranes during virus entry. Naturally occurring neutralizing antibodies against herpesviruses target these entry proteins. To determine the molecular basis for VZV neutralization, crystal structures of gHgL were determined in complex with fragments of antigen binding (Fabs) from two human monoclonal antibodies, IgG-94 and IgG-RC, isolated from seropositive subjects. These structures reveal that the antibodies target the same site, composed of residues from both gH and gL, distinct from two other neutralizing epitopes identified by negative-stain electron microscopy and mutational analysis. Inhibition of gB/gHgL-mediated membrane fusion and structural comparisons with herpesvirus homologs suggest that the IgG-RC/94 epitope is in proximity to the site on VZV gHgL that activates gB. Immunization studies proved that the anti-gHgL IgG-RC/94 epitope is a critical target for antibodies that neutralize VZV. Thus, the gHgL/Fab structures delineate a site of herpesvirus vulnerability targeted by natural immunity.

Mutations in the GLA Gene and LysoGb3: Is It Really Anderson-Fabry Disease?

Anderson-Fabry disease (FD) is a rare, progressive, multisystem storage disorder caused by the partial or total deficit of the lysosomal enzyme α-galactosidase A (α-Gal A). It is an X-linked, lysosomal enzymopathy due to mutations in the galactosidase alpha gene (GLA), encoding the α-Gal A. To date, more than 900 mutations in this gene have been described. In our laboratories, the study of genetic and enzymatic alterations related to FD was performed in about 17,000 subjects with a symptomatology referable to this disorder. The accumulation of globotriaosylsphingosine (LysoGb3) was determined in blood of positives. Exonic mutations in the GLA gene were detected in 471 patients (207 Probands and 264 relatives): 71.6% of mutations were associated with the classic phenotype, 19.8% were associated with the late-onset phenotype, and 8.6% of genetic variants were of unknown significance (GVUS). The accumulation of LysoGb3 was found in all male patients with a mutation responsible for classic or late-onset FD. LysoGb3 levels were consistent with the type of mutations and the symptomatology of patients. α-Gal A activity in these patients is absent or dramatically reduced. In recent years, confusion about the pathogenicity of some mutations led to an association between non-causative mutations and FD. Our study shows that the identification of FD patients is possible by associating clinical history, GLA gene analysis, α-Gal A assay, and blood accumulation of LysoGB3. In our experience, LysoGB3 can be considered a reliable marker, which is very useful to confirm the diagnosis of Fabry disease.

Antigenic Characterization of the HCMV gH/gL/gO and Pentamer Cell Entry Complexes Reveals Binding Sites for Potently Neutralizing Human Antibodies.

Human Cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and in fetuses following congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are required for HCMV entry in fibroblasts and endothelial/epithelial cells, respectively, and are targeted by potently neutralizing antibodies in the infected host. Using purified soluble forms of gH/gL/gO and Pentamer as well as a panel of naturally elicited human monoclonal antibodies, we determined the location of key neutralizing epitopes on the gH/gL/gO and Pentamer surfaces. Mass Spectrometry (MS) coupled to Chemical Crosslinking or to Hydrogen Deuterium Exchange was used to define residues that are either in proximity or part of neutralizing epitopes on the glycoprotein complexes. We also determined the molecular architecture of the gH/gL/gO- and Pentamer-antibody complexes by Electron Microscopy (EM) and 3D reconstructions. The EM analysis revealed that the Pentamer specific neutralizing antibodies bind to two opposite surfaces of the complex, suggesting that they may neutralize infection by different mechanisms. Together, our data identify the location of neutralizing antibodies binding sites on the gH/gL/gO and Pentamer complexes and provide a framework for the development of antibodies and vaccines against HCMV.

Development of a degenerated TaqMan real-time Q-PCR for detection of bacteria-free DNA in dialysis fluid.

Bacterial-derived DNA fragments (BDNAs) have been shown to be present in a dialysis fluid, to pass through dialyzer membranes, and to induce interleukin 6 (IL-6) in mononuclear cells. DNA fragments are thought to be derived from microorganisms inhabiting hemodialysis water and fluid. The primary aim of the present study was to develop two degenerated TaqMan real-time quantitative-PCR (Q-PCR) for detection of a broad range of bacterial DNA that specifically detect 16S ribosomal DNA (rDNA) (862 and 241 bp) and evaluate the efficiency of the Bellco Selecta resin to capture the BDNAs in the dialysis fluid. For this purpose, we decided to compare measurements of unfragmented samples (9.8 × 105 Escherichia coli genome) with artificially fragmented DNA samples. We assessed two broad-range real-time PCR targeting bacterial 16S rRNA genes for detection of fragmented and unfragmented bacterial DNA in the dialytic fluid and demonstrated that Bellco Selecta resin is capable of retaining these types of bacterial DNA.

Vitamins (A, C and E) and oxidative status of hemodialysis patients treated with HFR and HFR-Supra.

BACKGROUND: Hemodiafiltration with on-line endogenous reinfusion (HFR) is an extracorporeal dialytic method that combines diffusion, convection and adsorption. HFR-Supra (HFR-S) is a second-generation system with increased convective permeability and adsorption capability. Previous studies suggested that HFR reduces oxidative stress compared to standard haemodialysis. The principal aim of the present study was to compare antioxidant vitamins behavior and oxidative status of hemodialysis patients treated with HFR and HFR-S. METHODS: The study was designed as a multicenter, randomized, crossover trial. Forty-one patients were recruited from 19 dialysis centers and after a 4-month washout stabilization period in on-line hemodiafiltration (ol-HDF), each patient was randomized to a sequence of treatments (HFR-S followed by HFR or viceversa) with each treatment applied over 6 months. Plasma levels of Advanced Oxidation Protein Products, Total Antioxidant Status, vitamins C, A and E and their ligands (Retinol Binding Protein and total lipids) were measured at baseline and at the end of each treatment period. RESULTS: Results show that the higher convective permeability of HFR-S with respect to HFR did not produce additional beneficial effects on the patients' oxidative status, a slight decrease of both Vitamin A and Retinol Binding Protein being the only difference registered in the long-term. However, as compared to ol-HDF, both the re-infusive techniques allowed to reduce the intradialytic loss of Vitamin C and, in the long-term, improve the patients' oxidative status and increase Retinol Binding Protein plasma values. No significant differences were found between the Vitamin C concentration of pre- and post cartridge UF neither in HFR-S nor in HFR showing that the sorbent resin does not adsorb Vitamin C. CONCLUSION: HFR-S and HFR are almost equivalent in term of impact on antioxidant vitamins and oxidative status of hemodialysis patients. Nonetheless, as compared to ol-HDF, both treatments produced a sensible sparing of Vitamin C and may represent a new approach for reducing oxidative stress and related complications in dialysis patients. Long-term effects of re-infusive treatments on patients' cardiovascular morbidity and mortality need to be evaluated. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT01492491 , retrospectively registered in 10 December 2011.

Circulating adiponectin and cardiovascular mortality in patients with type 2 diabetes mellitus: evidence of sexual dimorphism.

BACKGROUND: The pathogenesis of cardiovascular (CV) mortality, whose rate is increased in type 2 diabetes, is poorly understood. METHODS: Three prospective cohorts were analyzed: 1) Gargano Heart Study (GHS; 359 patients, 58 events/1,934 person-years; py); 2) Health Professional Follow-up Study (HPFS; 833 men, 146 events/10,024 py); 3) Nurses' Health Study (NHS; 902 women, 144 events/15,074 py). RESULTS: In GHS serum adiponectin predicted CV mortality in men (hazard ratio, HR, and 95% CI per standard deviation, SD, increment = 1.54, 1.19-2.01), but not women (HR = 0.98, 0.48-2.01). CONCLUSIONS: This is the first report showing that high circulating adiponectin predicts increased CV mortality in men, but not in women with type 2 diabetes. Further studies are necessary to unravel the mechanisms through which adiponectin influences CV mortality in a sex-specific manner.

Proteomic analisys of protein extraction during hemofiltration with on-line endogenous reinfusion (HFR) using different polysulphone membranes.

In end-stage renal disease patients, extracorporeal dialytic therapy is not able to prevent the accumulation of toxins related to the uremic syndrome, a severe complication that increases morbidity and mortality rate. In this paper, hemoFiltration with on-line Reinfusion (HFR) architecture is used to evaluate the effect of a more permeable membrane on the extraction of medium-high molecular weight molecules. The aim of this study was to compare two polysulphone membranes for convective chamber: polyphenylene High Flux (pHF) and polyphenylene Super High-Flux (pSHF). Fourteen patients were subjected to HFR with pHF and pSHF membranes and ultra filtrate (UF) samples were collected to evaluate molecular weight cut-off (MWCO) and to identify extracted proteins. Furthermore, image analysis software was used in order to evaluate change in protein extraction during the dialysis. The quantification of four proteins by immunoassay demonstrates a higher permeability of pSHF membrane. Two-dimensional electrophoresis (2-DE) gels showed, for both membranes, the greater number of protein spots at 235 min. Some of the identified proteins, involved in nephropathic disease complications, were compared to assess differences in extraction during dialytic treatment by PDQuest analysis. UF proteomic analysis demonstrated a different behavior for the two membranes; pHF membrane was more permeable at the beginning of HFR treatment (15 min), while pSHF membrane at the end of treatment (235 min). Proteomic analysis is a suitable approach to investigate the behavior of different membranes during dialysis. Results indicated that pSHF membrane offers the higher permeability, and showed higher efficiency in removal of middle molecules related to uremic syndrome.

Erratum: Self-amplifying mRNA bicistronic influenza vaccines raise cross-reactive immune responses in mice and prevent infection in ferrets.

[This corrects the article DOI: 10.1016/j.omtm.2022.09.013.].

Self-amplifying mRNA seasonal influenza vaccines elicit mouse neutralizing antibody and cell-mediated immunity and protect ferrets.

Currently licensed influenza vaccines focus immune responses on viral hemagglutinin (HA), while the other major surface glycoprotein neuraminidase (NA) is not tightly controlled in inactivated vaccine formulations despite evidence that anti-NA antibodies reduce clinical disease. We utilized a bicistronic self-amplifying mRNA (sa-mRNA) platform encoding both HA and NA from four seasonal influenza strains, creating a quadrivalent influenza vaccine. sa-mRNA vaccines encoding an NA component induced the production of NA-inhibiting antibodies and CD4+ T-cell responses in both monovalent and quadrivalent formulations. Including NA in the vaccine enabled cross-neutralization against antigenically drifted strains and provided greater protection than HA alone upon A(H3N2) challenge in ferrets. These results demonstrate that next-generation bicistronic sa-mRNA vaccines expressing HA and NA induce potent antibodies against both viral coat proteins, as well as vaccine-specific cell-mediated immunity. When formulated as a quadrivalent seasonal influenza vaccine, the sa-mRNA platform provides an opportunity to increase the breadth of protection through cross-neutralizing anti-NA antibodies.

Assessing the stability-indicating properties of alternative potency assays for inactivated influenza vaccine.

Determination of the potency of a vaccine is critical to ensuring that an appropriate dose is delivered, lot-to-lot consistency is maintained, and that the formulation is stable over the life of the vaccine. The potency of inactivated influenza vaccines is determined routinely by the Single Radial Immunodiffusion (SRID) assay. A number of alternative potency assays have been proposed and have been under evaluation in recent years. The aim of this study was to compare a surface plasmon resonance-based assay and two different enzyme linked immunoassays against the current potency assay, SRID, and against mouse immunogenicity when haemagglutinin antigen of the A(H1N1)pdm09 component of an inactivated influenza vaccine is stressed by elevated temperature, low pH and freezing. This analysis demonstrated that the alternative assays had good correspondence with SRID for samples from most stress conditions and that the immunogenicity in mice corresponded with potency in SRID for all stress samples. Subject to further analysis, the assays have been shown to have the potential to possibly replace, and at least complement, SRID.

Haemagglutination inhibition and virus microneutralisation serology assays: use of harmonised protocols and biological standards in seasonal influenza serology testing and their impact on inter-laboratory variation and assay correlation: A FLUCOP collaborative study.

Introduction: The haemagglutination inhibition assay (HAI) and the virus microneutralisation assay (MN) are long-established methods for quantifying antibodies against influenza viruses. Despite their widespread use, both assays require standardisation to improve inter-laboratory agreement in testing. The FLUCOP consortium aims to develop a toolbox of standardised serology assays for seasonal influenza. Building upon previous collaborative studies to harmonise the HAI, in this study the FLUCOP consortium carried out a head-to-head comparison of harmonised HAI and MN protocols to better understand the relationship between HAI and MN titres, and the impact of assay harmonisation and standardisation on inter-laboratory variability and agreement between these methods. Methods: In this paper, we present two large international collaborative studies testing harmonised HAI and MN protocols across 10 participating laboratories. In the first, we expanded on previously published work, carrying out HAI testing using egg and cell isolated and propagated wild-type (WT) viruses in addition to high-growth reassortants typically used influenza vaccines strains using HAI. In the second we tested two MN protocols: an overnight ELISA-based format and a 3-5 day format, using reassortant viruses and a WT H3N2 cell isolated virus. As serum panels tested in both studies included many overlapping samples, we were able to look at the correlation of HAI and MN titres across different methods and for different influenza subtypes. Results: We showed that the overnight ELISA and 3-5 day MN formats are not comparable, with titre ratios varying across the dynamic range of the assay. However, the ELISA MN and HAI are comparable, and a conversion factor could possibly be calculated. In both studies, the impact of normalising using a study standard was investigated, and we showed that for almost every strain and assay format tested, normalisation significantly reduced inter-laboratory variation, supporting the continued development of antibody standards for seasonal influenza viruses. Normalisation had no impact on the correlation between overnight ELISA and 3-5 day MN formats.

Quantification of p-cresol sulphate in human plasma by selected reaction monitoring.

Chronic renal failure patients accumulate in the blood molecules that are normally excreted into the urine. p-Cresol Sulphate (pCS), the most representative retained toxin, shows a high level of toxicity. Therefore, its quantification could represent a prediction factor to determine the risk of endothelial dysfunction and cardiovascular complication and response to the haemodialysis treatment. The aim of this study was to evaluate the suitability of the multiple reaction monitoring (MRM) technique in order to improve the sensibility, the selectivity and the timing of pCS detection in a small amount of plasma. Deproteinized plasma of uremic patients was concentrated and dissolved in liquid chromatography (LC) mobile phase solution. pCS was quantified by LC coupled to tandem mass spectrometry (LC-MS/MS) on a triple-quadrupole mass spectrometer. Selective and sensitive detection of pCS was achieved by selecting the specific parent ion and monitoring two specific fragment ions. The MRM assay was carried out using the following transitions: m/z 187 → 80.00 and m/z 187 → 107.00. A good linearity was observed for each calibration curve. The intra-day and inter-day results showed a good precision and repeatability. The percentage recoveries indicate an optimal selectivity of the analytical method. The MRM assay to quantify pCS in a small amount of human plasma is rapid, highly sensitive, selective and with a good repeatability.

Self-amplifying mRNA SARS-CoV-2 vaccines raise cross-reactive immune response to variants and prevent infection in animal models.

The spike (S) protein of SARS-CoV-2 plays a crucial role in cell entry, and the nucleocapsid (N) protein is highly conserved among human coronavirus homologs. For potentially broad effectiveness against both original virus and emerging variants, we developed Alphavirus-based self-amplifying mRNA (sa-mRNA) SARS-CoV-2 vaccines: an sa-mRNA S encoding a full-length S protein stabilized in a prefusion conformation and an sa-mRNA S-N co-expressing S and N proteins for the original virus. We show that these sa-mRNA SARS-CoV-2 vaccines raised potent neutralizing antibody responses in mice against not only the original virus but also the Alpha, Beta, Gamma, and Delta variants. sa-mRNA S vaccines against the Alpha and Beta variants also raised robust cross-reactive neutralizing antibody responses against their homologous viruses and heterologous variants. sa-mRNA S and sa-mRNA S-N vaccines elicited Th1-dominant, antigen-specific CD4+ T cell responses to S and N proteins and robust and broad CD8+ T cell responses to S protein. Hamsters immunized with either vaccine were fully protected from lung infection and showed significant reduction of viral load in upper respiratory tract. Our findings demonstrate that sa-mRNA SARS-CoV-2 vaccines are potent in animal models with potential to be highly effective against SARS-CoV-2 infection in humans.

Self-amplifying mRNA bicistronic influenza vaccines raise cross-reactive immune responses in mice and prevent infection in ferrets.

Vaccines are the primary intervention against influenza. Currently licensed inactivated vaccines focus immunity on viral hemagglutinin (HA). Self-amplifying mRNA (sa-mRNA) vaccines offer an opportunity to generate immunity to multiple viral proteins, including additional neuraminidase (NA). This evaluation of a bicistronic approach for sa-mRNA vaccine development compared subgenomic promoter and internal ribosome entry site strategies and found consistent and balanced expression of both HA and NA proteins in transfected cells. In mice, sa-mRNA bicistronic A/H5N1 vaccines raised potent anti-HA and anti-NA neutralizing antibody responses and HA- or NA-specific CD4+ and CD8+ T cell responses. The addition of NA also boosted the cross-neutralizing response to heterologous A/H1N1. Similar immunogenicity results were obtained for bicistronic seasonal A/H3N2 and B/Yamagata vaccines. In ferrets, sa-mRNA bicistronic A/H1N1 vaccine fully protected lung from infection by homologous virus and showed significant reduction of viral load in upper respiratory tract, warranting further evaluation of sa-mRNA bicistronic vaccine in humans.