Humoral and cellular immune responses after 6 months of a heterologous SARS-CoV-2 booster with the protein-based PHH-1V vaccine in a phase IIb trial (preprint)

The HIPRA-HH-2 was a multicentre, randomized, active-controlled, double-blind, non-inferiority phase IIb clinical trial to compare the immunogenicity and safety of a heterologous booster with PHH-1V adjuvanted recombinant vaccine versus a homologous booster with mRNA vaccine. Interim results showed a strong humoral and cellular immune response against the SARS-CoV-2 Wuhan-Hu-1 strain and the Beta, Delta, and Omicron BA.1 variants up to day 98 after dosing. Here we report that these humoral and cellular responses after PHH-1V dosing are sustained up to 6 months. These results are observed both when including or not participants who reported SARS-CoV-2 infection and in a high-risk population ([≥]65 years). Additional analysis revealed a non-inferiority of PHH-1V booster in eliciting neutralizing antibodies also for SARS-CoV-2 Omicron XBB.1.5 when compared to mRNA vaccine after 6 months. The PHH-1V vaccine provides long-lasting protection against a wide variety of SARS-CoV-2 emerging variants to prevent severe COVID-19. ClinicalTrials.gov Identifier: NCT05142553

Novel Spike-stabilized trimers with improved production protect K18-hACE2 mice and golden Syrian hamsters from the highly pathogenic SARS-CoV-2 Beta variant. (preprint)

Most COVID-19 vaccines are based on the SARS-CoV-2 Spike glycoprotein (S) or their subunits. However, the S shows some structural instability that limits its immunogenicity and production, hampering the development of recombinant S-based vaccines. The introduction of the K986P and V987P (S-2P) mutations increases the production of the recombinant S trimer and, more importantly, its immunogenicity, suggesting that these two parameters are related. However, S-2P still shows some molecular instability and it is produced with low yield. Thus, S-2P production can be further optimized. Here we described a novel set of mutations identified by molecular modelling and located in the S2 region of the Spike that increase S-2P production up to five-fold. Besides their immunogenicity, the efficacy of two representative S-2P-based mutants, S-29 and S-21, protecting from a heterologous SARS-CoV-2 Beta variant challenge was assayed in K18-hACE2 mice (an animal model of severe SARS-CoV-2 disease) and golden Syrian hamsters (GSH) (a moderate disease model). S-21 induced higher level of WH1 and Delta variants neutralizing antibodies than S-2P in K18-hACE2 mice three days after challenge. Viral load in nasal turbinate and oropharyngeal samples were reduced in S-21 and S-29 vaccinated mice. Despite that, only the S-29 protein protected 100% of K18-hACE2 mice from severe disease. When GSH were analyzed, all immunized animals were protected from disease development irrespectively of the immunogen they received. Therefore, the higher yield of S-29, as well as its improved immunogenicity and efficacy protecting from the highly pathogenic SARS-CoV-2 Beta variant, pinpoint the S-29 spike mutant as an alternative to the S-2P protein for future SARS-CoV-2 vaccine development.

Immunization with V987H-stabilized Spike glycoprotein protects K18-hACE2 and golden Syrian hamster upon SARS-CoV-2 infection. (preprint)

Safe and effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have been crucial to fight against the coronavirus disease 2019 pandemic. Most vaccines are based on a mutated version of the Spike glycoprotein [K986P/V987P (S-2P)] with improved stability, yield and immunogenicity. However, S-2P is still produced at low levels. Here, we described a novel V987H mutation that increases by two-fold the production of the recombinant Spike and the exposure of the receptor binding domain (RBD). S-V987H immunogenicity was similar to S-2P in K18-hACE2 mice and golden Syrian hamsters, and superior to a monomeric RBD. Immunization with S-V987H, but not with S-2P or RBD, conferred full protection against severe disease in both animal models after SARS-CoV-2 challenge (D614G and B.1.351 variants). Furthermore, S-V987H immunized K18-hACE2 mice showed a faster tissue viral clearance than RBD- or S-2P-vaccinated animals. Thus, S-V987H protein provides an alternative to S-2P for future SARS-CoV-2 vaccines development.

Beta-Cyclodextrins as affordable antivirals to treat coronavirus infection (preprint)

The SARS-CoV-2 pandemic made evident that we count with few coronavirus-fighting drugs. Here we aimed to identify a cost-effective antiviral with broad spectrum activity and high safety and tolerability profiles. We began elaborating a list of 116 drugs previously used to treat other pathologies or characterized in pre-clinical studies with potential to treat coronavirus infections. We next employed molecular modelling tools to rank the 44 most promising inhibitors and tested their efficacy as antivirals against a panel of alpha and beta coronavirus, e.g., the HCoV-229E and SARS-CoV-2 viruses. Four drugs, OSW-1, U18666A, hydroxypropyl-beta-cyclodextrin (HbetaCD) and phytol, showed antiviral activity against both HCoV-229E (in MRC5 cells) and SARS-CoV-2 (in Vero E6 cells). The mechanism of action of these compounds was studied by transmission electron microscopy (TEM) and by testing their capacity to inhibit the entry of SARS-CoV-2 pseudoviruses in ACE2-expressing HEK-293T cells. The entry was inhibited by HbetaCD and U18666A, yet only HbetaCD could inhibit SARS-CoV-2 replication in the pulmonary cells Calu-3. With these results and given that cyclodextrins are widely used for drug encapsulation and can be safely administered to humans, we further tested 6 native and modified cyclodextrins, which confirmed {beta}-cyclodextrins as the most potent inhibitors of SARS-CoV-2 replication in Calu-3 cells. All accumulated data points to beta-cyclodextrins as promising candidates to be used in the therapeutic treatments for SARS-CoV-2 and possibly other respiratory viruses.

Safety and Immunogenicity of a Recombinant Protein RBD Fusion Heterodimer Vaccine against SARS-CoV-2: preliminary results of a phase 1-2a dose-escalating, randomized, double-blind clinical trial. (preprint)

Summary Background: We report safety, tolerability, and immunogenicity of a recombinant protein RBD-fusion heterodimeric vaccine against SARS-CoV-2 (PHH-1V). Methods: A dose-escalation, phase 1-2a, randomized clinical trial was performed in Catalonia, Spain. Each cohort had one safety sentinel that received PHH-1V vaccine of the corresponding dose, and remaining participants were randomly assigned to receive PHH-1V formulations [10mcg (n=5), 20mcg (n=10), 40mcg (n=10)] or control BNT162b2 (n=5). Two intramuscular doses (0-21 days) were administered. Primary endpoint was solicited events 7 days after each vaccination and secondary-exploratory endpoints were humoral and cellular immunogenicity. Findings: 30 young healthy adults were enrolled, thirteen were female. Vaccines were safe, well tolerated. The most common solicited events for all groups were tenderness and pain at the site of injection. The proportion of subjects with at least one reported local and/or systemic solicited adverse events (AE) after first or second vaccine dose were lowest in PHH-1V (n=21, 84%) than control group (n=5, 100%). AE were mild to moderate, and no severe AE nor AE of special interest were reported. All participants had a >4-fold change at day 35 in total binding antibodies from baseline. Variants of concern (VOC) alpha, beta, delta and gamma were evaluated using a SARS-CoV-2 pseudovirus-based neutralization assay. All groups had a significant geometric mean fold rise (p

Safety and immunogenicity of the protein-based PHH-IV compared to BNT162b2 as a heterologous SARS-CoV-2 booster vaccine in adults vaccinated against COVID-19: a multicentre, randomised, double-blind, non-inferiority phase IIb trial (preprint)

Summary Background A SARS-CoV-2 protein-based heterodimer vaccine, PHH-1V, has been shown to be safe and well-tolerated in healthy young adults in a first-in-human, Phase I/IIa study dose-escalation trial. Here, we report the interim results of the Phase IIb HH-2, where the immunogenicity and safety of a heterologous booster with PHH-1V is assessed versus a homologous booster with BNT162b2 at 14 and 98 days after vaccine administration. Methods The HH-2 study is an ongoing multicentre, randomised, active-controlled, double-blind, non-inferiority Phase IIb trial, where participants 18 years or older who had received two doses of BNT162b2 were randomly assigned in a 2:1 ratio to receive a booster dose of vaccine – either heterologous (PHH-1V group) or homologous (BNT162b2 group) – in 10 centres in Spain. Eligible subjects were allocated to treatment stratified by age group (18-64 versus ≥65 years) with approximately 10% of the sample enrolled in the older age group. The endpoints were humoral immunogenicity measured by changes in levels of neutralizing antibodies against the ancestral Wuhan-Hu-1 strain and different variants of SARS-CoV-2 after the PHH-1V or the BNT162b2 boost, the T-cell responses towards the SARS-CoV-2 spike glycoprotein peptides and the safety and tolerability of PHH-1V as a boost. This study is ongoing and is registered with ClinicalTrials.gov, NCT05142553 . Findings From 15 November 2021, 782 adults were randomly assigned to PHH-1V (n=522) or BNT162b2 (n=260) boost vaccine groups. The geometric mean titre (GMT) ratio of neutralizing antibodies on days 14 and 98, shown as BNT162b2 active control versus PHH-1V, was, respectively, 1·68 (p<0·0001) and 0·87 (p=0·43) for the ancestral Wuhan-Hu-1 strain; 0·61 (p<0·0001) and 0·57 (p=0·0064) for the beta variant; 1·01 (p=0·89) and 0·52 (p=0·0003) for the delta variant; and 0·59 (p=<0·0001) and 0·56 (p=0·0026) for the omicron variant. Additionally, PHH-1V as a booster dose induced a significant increase of CD4 + and CD8 + T-cells expressing IFN-γ on day 14. There were 458 participants who experienced at least one adverse event (89·3%) in the PHH-1V and 238 (94·4%) in the BNT162b2 group. The most frequent adverse events were injection site pain (79·7% and 89·3%), fatigue (27·5% and 42·1%) and headache (31·2 and 40·1%) for the PHH-1V and the BNT162b2 groups, respectively. A total of 52 COVID-19 cases occurred from day 14 post-vaccination (10·14%) for the PHH-1V group and 30 (11·90%) for the BNT162b2 group (p=0·45), and none of the subjects developed severe COVID-19. Interpretation Our interim results from the Phase IIb HH-2 trial show that PHH-1V as a heterologous booster vaccine, when compared to BNT162b2, elicits a strong and sustained neutralizing antibody response against Wuhan-Hu-1 strain, and a superior one concerning the previous circulating beta and delta SARS-CoV-2 variants, as well as the currently circulating omicron. Moreover, the PHH-1V boost also induces a strong and balanced T-cell response. Concerning the safety profile, subjects in the PHH-1V group report significantly fewer adverse events than those in the BNT162b2 group, most of mild intensity, and both vaccine groups present comparable COVID-19 breakthrough cases, none of them severe. Funding HIPRA SCIENTIFIC, S.L.U.

Monitoring SARS-CoV-2 variant transitions using differences in diagnostic cycle threshold values of target genes (preprint)

Monitoring the emergence of new SARS-CoV-2 variants is important to detect potential risks of increased transmission or disease severity. We investigated the identification of SARS-CoV-2 variants from real-time reverse transcriptase polymerase chain reaction (RT-PCR) routine diagnostics data. Cycle threshold (Ct) values of positive samples were collected from April 2021 to January 2022 in the Northern Metropolitan Area of Barcelona (n = 15,254). Viral lineage identification from whole-genome sequencing (WGS) was available for 4,618 (30.3%) of these samples. Pairwise differences in the Ct values between gene targets (ΔCt) were analyzed for variants of concern or interest circulating in our area. A specific delay in the Ct of the N-gene compared to the RdRp-gene (ΔCtNR) was observed for Alpha, Delta, Eta and Omicron. Temporal differences in ΔCtNR correlated with the dynamics of viral replacement of Alpha by Delta and of Delta by Omicron according to WGS results. Using ΔCtNR, prediction of new variants of concern at early stages of circulation was achieved with high sensitivity and specificity (91.1% and 97.8% for Delta; 98.5% and 90.8% for Omicron). Thus, tracking population-wide trends in ΔCt values obtained from routine diagnostics testing in combination with WGS could be useful for real-time management and response to local epidemics.

Kinetics of immune responses elicited after three mRNA COVID-19 vaccine doses in predominantly antibody-deficient individuals. (preprint)

Purpose Mass vaccination campaigns have reduced the incidence and severity of COVID-19. However, there is limited information about how patients with predominantly antibody-deficiencies (PAD) respond to COVID-19 vaccination. Here, we evaluated humoral and cellular responses developed in SARS-CoV-2-naïve PAD individuals after three mRNA-1273 vaccine doses.Methods Patients and healthy controls (HCs) were immunized at week 0 (w0) and w4. PAD individuals received an additional dose at w24. Blood samples were collected at w0, w4, w8, w24, and/or w28. We determined levels of anti-Spike and anti-RBD antibodies, Spike-specific IgG avidity, and neutralizing activity (Wuhan-Hu-1, Delta, and Omicron variants). Cellular responses were evaluated by IFN-γ ELISpot and flow cytometry.Results Unclassified primary antibody-deficiency patients (unPAD, n = 9) and HCs developed comparable vaccine-induced humoral responses. However, common variable immunodeficiency patients (CVID, n = 12) showed lower antibody responses than HCs. While the frequency of Spike-specific CD4 + T cells was similar between PAD patients and HCs, CD8 + T cells responses were reduced in CVID individuals. Both PAD groups showed lower levels of Spike-specific IFN-γ-producing T-cells. Combined immunodeficiency (CID, n = 1) and thymoma with immunodeficiency (TID, n = 1) patients developed cellular but not humoral responses after two immunizations. The third vaccine dose boosted humoral responses in most PAD patients, but had little effect on cellular immunity.Conclusion mRNA-1273 vaccine-induced immune responses in PAD individuals are heterogeneous, depend on the type and degree of antibody-deficiency, and should be immunomonitored to define a personalized vaccination strategy.

Kinetics of humoral immune response over 17 months of COVID-19 pandemic in a large cohort of healthcare workers in Spain: The ProHEpiC-19 study (preprint)

BackgroundUnderstanding the immune response to the SARS-CoV-2 virus is critical for efficient monitoring and defence strategies. The ProHEpic-19 cohort provides a fine-grained description of the kinetics of antibodies after SARS-CoV-2 infection with an exceptional resolution over 17 months.MethodsWe established a cohort of 769 health professionals including healthy and infected with SARS-CoV-2 in northern Barcelona to determine the kinetics of the IgM against the nucleocapsid (N) and the IgG against the N and spike (S) of SARS-CoV-2. We used non-linear mixed models to investigate the kinetics of IgG and IgM measured at nine time points over 17 months from the diagnosis. The model included factors of time, gender, and disease severity (asymptomatic, mild-moderate, severe-critical) to assess their effects and their interactions.Findings: 474 of the 769 participants (61.6%) became infected with SARS-CoV-2. Significant effects of gender and disease severity were found for the levels of all three antibodies. Median IgM(N) levels were already below the positivity threshold in patients with asymptomatic and mild-moderate disease at day 270 after the diagnosis, while IgG(N and S) levels remained positive at least until days 450 and 270, respectively. Kinetic modelling showed a general rise in both IgM(N) and IgG(N) levels up to day 30, followed by a decay with a rate depending on disease severity. IgG(S) levels remained relatively constant from day 15 over time.Interpretation: IgM(N) and IgG(N, S) SARS-CoV-2 antibodies showed a heterogeneous kinetics over the 13 months. Only the IgG(S) showed a stable increase, and the levels and the kinetics of antibodies varied according to disease severity. The kinetics of IgM and IgG observed over a year also varied by clinical spectrum can be very useful for public health policies around vaccination criteria in adult population.Funding: Regional Ministry of Health of the Generalitat de Catalunya (Call COVID19-PoC SLT16_04; NCT04885478) 

Comparison between mid-nasal swabs and buccal swabs for SARS-CoV-2 detection in mild COVID-19 patients (preprint)

Background The use of rapid antigen diagnostics tests (Ag-RDT) has gained widespread acceptance as an alternative method for diagnosis of COVID-19 outside of health care settings. Various authors have reported that saliva is a reliable specimen, alternative to nasopharyngeal and mid-nasal swabs, to detect SARS-CoV-2 infections by RT-PCR. We assessed the performance of buccal swabs containing saliva for SARS-CoV-2 detection by Ag-RDT, using mid-nasal specimens as a reference in the northern area of Barcelona (Catalonia, Spain) Methods In the context of routine clinical diagnosis of mild COVID-19 patients, we enrolled 300 adults in a study to directly compare mid-nasal swabs and saliva specimens for SARS-CoV-2 detection by Ag-RDT. When mid-nasal and buccal Ag-RDTs showed discordant results, a third mid-nasal swab was collected and analysed by RT-PCR. Results Paired samples were successfully obtained in 300 suspected cases of SARS-CoV-2 infection. Of the 300 paired samples, Ag-RDT with the mid-nasal swab detected 139 (46.3%) positive COVID-19 cases. In comparison, buccal swabs showed a sensitivity and specificity of 31.7% (44/139) and 98.8% (159/161), respectively. 65 discordant results with positive mid-nasal swabs and negative buccal swabs were tested by RT-qPCR. All samples tested by Rt-PCR resulted positive, with a mean cycle threshold (Ct) of 28.3 (SD 7.3). Conclusion Our findings show that mid-nasal swabs have better performance than buccal swabs for detecting SARS-CoV-2 with Ag-RDT tests. Of note, the sensitivity of buccal samples was affected in samples with high viral loads (Ct<33), suggesting that buccal swabs might not be sensitive enough to detect individuals at risk of transmission. Taken together, the existing literature and the results provided in our analysis we advise against the use of buccal specimens for SARS-CoV-2 diagnostics with Ag-RDT.

Cross-sectional study to assess the efficacy of SARS-CoV-2 vaccination in patients with monoclonal gammopathies (preprint)

SARS-CoV-2 vaccination is the most effective strategy to protect patients with haematologic malignancies against severe COVID-19, but primary vaccine responses are less effective in this population. Here, we characterized the humoral responses following 3 months after mRNA-based vaccines in patients at different stages of the same plasma cell diseases, including monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM) and multiple myeloma on first line therapy (MM), compared to a healthy control population matched by sex and age. We observed that plasmas from uninfected MM patients after 3 months post-vaccine have lower SARS-CoV-2 specific IgG and IgA antibodies and decreased neutralization capacity compared with MGUS and SMM individuals, and a group of healthy controls. Importantly, we detected significantly higher plasma neutralization capacity in MM individuals who recovered from COVID-19 compared to their uninfected counterparts, highlighting that hybrid immunity elicit stronger immune responses even in this immunocompromised population. In contrast to MM group, no differences in the vaccine-induced humoral response were observed between uninfected MGUS, SMM and healthy individuals. In conclusion, a booster vaccine dose is recommended in uninfected MM patients to develop an adequate and effective humoral response to SARS-CoV-2 vaccine.

Unraveling the antiviral activity of plitidepsin by subcellular and morphological analysis (preprint)

The pandemic caused by the new coronavirus SARS-CoV-2 has made evident the need for broad-spectrum, efficient antiviral treatments to combat emerging and re-emerging viruses. Plitidepsin is an antitumor agent of marine origin that has also shown a potent pre-clinical efficacy against SARS-CoV-2. Plitidepsin targets the host protein eEF1A (eukaryotic translation factor 1 alpha 1) and affects viral infection at an early, post-entry step. Because electron microscopy is a valuable tool to study virus-cell interactions and the mechanism of action of antiviral drugs, in this work we have used transmission electron microscopy (TEM) to evaluate the effects of plitidepsin in SARS-CoV-2 infection in cultured Vero E6 cells 24 and 48h post-infection. In the absence of plitidepsin, TEM morphological analysis showed double-membrane vesicles (DMVs), organelles that support coronavirus genome replication, single-membrane vesicles with viral particles, large vacuoles with groups of viruses and numerous extracellular virions attached to the plasma membrane. When treated with plitidepsin, no viral structures were found in SARS-CoV-2-infected Vero E6 cells. Immunogold detection of SARS-CoV-2 nucleocapsid (N) protein and double-stranded RNA (dsRNA) provided clear signals in cells infected in the absence of plitidepsin, but complete absence in cells infected and treated with plitidepsin. The present study shows that plitidepsin completely blocks the biogenesis of viral replication organelles and the morphogenesis of virus progeny. Electron microscopy morphological analysis coupled to immunogold labeling of SARS-CoV-2 products offers a unique approach to understand how antivirals such as plitidepsin work.

Convalescent plasma for outpatients with early COVID-19 (preprint)

Background: Convalescent plasma (CP) for hospitalized patients with COVID-19 has not demonstrated clear benefits. However, data on outpatients with early symptoms are scarce. We aimed to assess whether treatment with CP administered during the first 7 days of symptoms reduced the disease progression or risk of hospitalization of outpatients. Methods: Two double-blind randomized trials (NCT04621123, NCT04589949) were merged with data pooling starting when <20% of their predefined sample size had been recruited. A Bayesian adaptive individual patient data meta-analysis was implemented. Analyses were done with Bayesian proportional odds and logistic models, where odds ratios (OR)<1.0 indicate a favorable outcome for CP. Fourteen study sites across the Netherlands and Catalonia in Spain participated in the trial. The two studies included outpatients aged [≥]50 years and diagnosed with COVID-19 and symptomatic for [≤]7days. The intervention consisted of one unit (200-300mL) of CP with a predefined minimum level of antibodies. The two primary endpoints were (a) a 5-point disease severity scale (fully recovered by day 7 or not, hospital or ICU admission and death) and (b) a composite of hospitalization or death. Results: Of 797 patients included, 390 received CP and 392 placebo. At baseline, they had a median age of 58 years, 1 comorbidity, symptoms for 5 days and 93% tested negative for SARS-CoV-2 S-protein IgG antibodies. Seventy-four patients were hospitalized, 6 required mechanical ventilation and 3 died. The OR of CP for an improved disease severity scale was 0.936 (credible interval (CI) 0.667-1.311). The OR for hospitalization or death was 0.919 (CI 0.592-1.416). The effect of CP on hospital admission or death was largest in patients with [≤]5 days of symptoms (OR 0.658, 95% CI 0.394-1.085). CP did not decrease the time to full symptom resolution (p=0.62). Conclusion: Treatment with CP of outpatients in the first 7 days of symptoms did not improve the outcome of COVID-19. The possible beneficial effect in patients with [≤]5 days of symptoms requires further study. Registration: NCT04621123 and NCT04589949 on https://www.clinicaltrials.gov

A Cost-Benefit Analysis of COVID-19 Vaccination in Catalonia (preprint)

(1) Background: in epidemiological terms, it has been possible to calculate the savings in health resources and the reduction in health effects of COVID vaccines. From the point of view of economic evaluation, some studies have estimated its cost-effectiveness with the vaccination showing highly favorable results, which in some cases is cost-saving; (2) Methods: a cost-benefit analysis of the vaccination campaign in the North Metropolitan Health Region (Catalonia). An epidemiological model based on observational data and before and after comparison is used. The information on the doses used and the resources assigned (conventional hospital beds, ICU, number of tests) has been extracted from administrative data from the largest Primary Care provider in the region (Catalan Institute of Health). A distinction is made between the social perspective and the health system; (3) Results: the costs of vaccination are estimated at 137 million euros (€48.05/dose administered). This figure is significantly lower than the positive impacts of the vaccination campaign, which are estimated at 470 million euros (€164/dose administered). Of these, 18% corresponds to the reduction of ICU discharges, 16% to the reduction in conventional hospital discharges, 5% to the reduction in PCR tests and 1% to the reduction of RAT tests. Monetization of deaths and cases with sequelae avoided account for 53% and 5% of total savings, respectively. The benefit/cost ratio is estimated at 3.4 from a social perspective and 1.2 from a health system perspective. The social benefits of vaccination are estimated at €116.67 per dose of vaccine given (€19.93 from the point of view of the health system); (4) Conclusions: the mass vaccination campaign against COVID is cost-saving. From a social perspective, most of these savings come from the monetization of the reduction in mortality and cases with sequelae, although the intervention is equally widely cost-effective from the point of view of the health system thanks to the reduction in the use of resources. It is concluded that, from an economic perspective, the vaccination campaign has high social returns.

Preclinical efficacy, safety, and immunogenicity of PHH-1V, a second-generation COVID-19 vaccine candidate based on a novel recombinant RBD fusion heterodimer of SARS-CoV-2. (preprint)

Since the genetic sequence of SARS-CoV-2 became available in January 2020, new vaccines have been developed at an unprecedented speed. The current vaccines have been directly associated with a decline in new infection rates, prevention of severe disease and an outstanding decrease in mortality rates. However, the pandemic is still far from being over. New Variants of Concern (VoCs) are continuously evolving. Thus, it is essential to develop accessible second-generation COVID-19 vaccines against known and future VoCs to mitigate the current pandemic. Here, we provide preclinical data showing the immunogenicity, efficacy, and safety results in mice of a receptor-binding domain (RBD)-based recombinant protein vaccine candidate (PHH-1V) which consists of a novel RBD fusion heterodimer containing the B.1.1.7 (alpha) and B.1.351 (beta) variants of SARS-CoV-2, formulated with an oil-based adjuvant equivalent to MF59C.1. BALB/c and K18-hACE2 mice were immunized with different doses of recombinant RBD fusion heterodimer, following a two-dose prime-and-boost schedule. Upon 20 g RBD fusion heterodimer/dose immunization, BALB/c mice produced RBD-binding antibodies with neutralising activity against the alpha, beta, gamma, and delta variants. Furthermore, vaccination elicited robust activation of CD4+ and CD8+ T cells with early expression of Th1 cytokines upon in vitro restimulation, along with a good tolerability profile. Importantly, vaccination with 10 g or 20 g RBD fusion heterodimer/dose conferred 100% efficacy preventing mortality and bodyweight loss upon SARS-CoV-2 challenge in K18-hACE2 mice. These findings demonstrate the feasibility of this novel recombinant vaccine strategy, allowing the inclusion of up to 2 different RBD proteins in the same vaccine. Most importantly, this new platform is easy to adapt to future VoCs and has a good stability profile, thus ensuring its global distribution.

Limited immune responses after three months of BNT162b2 vaccine in SARS-CoV-2 uninfected elders living in long-term care facilities (preprint)

Background SARS-CoV-2 vaccination is the most effective strategy to protect elders living in long-term care facilities (LTCF) against severe COVID-19, but primary vaccine responses are less effective in older adults. Here, we characterized the humoral responses following 3 months after mRNA/BNT162b2 vaccine in institutionalized elders. Methods Plasma levels of specific SARS-CoV-2 total IgG, IgM and IgA antibodies were measured before and 3 months after vaccination in elders living in LTCF. Neutralization capacity was assessed in a pseudovirus neutralization assay against WH1 (original) and B.1.617.2/Delta variants. A group of younger adults was used as reference group. Results Three months after vaccination, uninfected-elders presented reduced specific SARS-CoV-2 IgG levels and significantly lower neutralization capacity against the WH1 and Delta virus compared to vaccinated uninfected younger individuals. In contrast, COVID-19 recovered elders showed significantly higher specific SARS-CoV-2 IgG levels after vaccination than younger counterparts, while showing similar neutralization activity against WH1 virus and increased neutralization capacity against Delta variant. Despite previously infected elders elicit potent cross-reactive immune responses similarly to younger individuals, higher quantities of specific SARS-CoV-2 IgG antibodies are required to reach the same neutralization levels. Conclusions While hybrid immunity seems to be active in previously infected elders after three months from mRNA/BNT162b2 vaccination, humoral immune responses are diminished in COVID-19 uninfected vaccinated residents living in LTCF. These results suggests that a vaccine booster dose should be prioritized for this particularly vulnerable population. Word summary While previously infected and vaccinated elders living in LTCF had comparable neutralizing antibody levels to younger individuals, vaccinated uninfected-residents showed limited neutralization capacity against both original and delta variants. Hybrid immunity seems to be active in elders and can be relevant to design vaccine boosting campaigns.

Antibody kinetics to SARS-CoV-2 at 13.5 months, by disease severity (preprint)

BackgroundUnderstanding humoral responses and seroprevalence in SARS-CoV-2 infection is essential for guiding vaccination strategies in both infected and uninfected individuals. MethodsWe determine the kinetics of IgM against the nucleocapsid (N) and IgG against the spike (S) and N proteins of SARS-CoV-2 in a cohort of 860 health professionals (healthy and infected) in northern Barcelona. We model the kinetics of IgG and IgM at nine time points over 13.5 months from infection, using non-linear mixed models by sex and clinical disease severity. ResultsOf the 781 participants who were followed up, 478 (61.2%) became infected with SARS-CoV-2. Significant differences were found for the three antibodies by disease severity and sex. At day 270 after diagnosis, median IgM(N) levels were already below the positivity threshold in patients with asymptomatic and mild-moderate disease, while IgG(N, S) levels remained positive to days 360 and 270, respectively. Kinetic modelling showed a general rise in both IgM(N) and IgG(N) levels up to day 30, followed by a decay whose rate depended on disease severity. IgG(S) levels increased at day 15 and remained relatively constant over time. ConclusionsWe describe kinetic models of IgM(N) and IgG(N, S) SARS-CoV-2 antibodies at 13.5 months from infection and disease spectrum. Our analyses delineate differences in the kinetics of IgM and IgG over a year and differences in the levels of IgM and IgG as early as 15 days from symptoms onset in severe cases. These results can inform public health policies around vaccination criteria. Funded by the regional Ministry of Health of the Generalitat de Catalunya (Call COVID19-PoC SLT16_04; NCT04885478)

Clinical course impacts early kinetics and long-term magnitude and amplitude of SARS-CoV-2 neutralizing antibodies beyond one year after infection (preprint)

Background Understanding the determinants of long-term immune responses to SARS-CoV-2 and the concurrent impact of vaccination and emerging variants of concern will guide optimal strategies to achieve global protection against the COVID-19 pandemic. Methods A prospective cohort of 332 COVID-19 patients was followed beyond one year. Plasma neutralizing activity was evaluated using HIV-based reporter pseudoviruses expressing different SARS-CoV-2 spikes and was longitudinally analyzed using mixed-effects models. Findings Long-term neutralizing activity was stable beyond one year after infection in mild/asymptomatic and hospitalized participants. However, longitudinal models suggest that hospitalized individuals generate both short- and long-lived memory B cells, while outpatient responses were dominated by long-lived B cells. In both groups, vaccination boosted responses to natural infection, although viral variants, mainly B.1.351, reduced the efficacy of neutralization. Importantly, despite showing higher neutralization titers, hospitalized patients showed lower cross-neutralization of B.1.351 variant compared to outpatients. Multivariate analysis identified severity of primary infection as the factor that independently determines both the magnitude and the inferior cross-neutralization activity of long-term neutralizing responses. Conclusions Neutralizing response induced by SARS-CoV-2 is heterogeneous in magnitude but stable beyond one year after infection. Vaccination boosts these long-lasting natural neutralizing responses, counteracting the significant resistance to neutralization of new viral variants. Severity of primary infection determines higher magnitude but poorer quality of long-term neutralizing responses.