Insights into the virologic and immunologic features of SARS-COV-2

The host immunity is crucial in determining the clinical course and prognosis of coronavirus disease 2019, where some systemic and severe manifestations are associated with excessive or suboptimal responses. Several antigenic epitopes in spike, nucleocapsid and membrane proteins of severe acute respiratory syndrome coronavirus 2 are targeted by the immune system, and a robust response with innate and adaptive components develops in infected individuals. High titer neutralizing antibodies and a balanced T cell response appears to constitute the optimal immune response to severe acute respiratory syndrome coronavirus 2, where innate and mucosal defenses also contribute significantly. Following exposure, immunological memory seems to develop and be maintained for substantial periods. Here, we provide an overview of the main aspects in antiviral immunity involving innate and adaptive responses with insights into virus structure, individual variations pertaining to disease severity as well as long-term protective immunity expected to be attained by vaccination.     

Clinical application of SARS-CoV-2 antibody detection and monoclonal antibody therapies against COVID-19

The purpose of this study was to investigate the clinical application of severe acute respiratory distress syndrome coronavirus-2(SARS-CoV-2) specific antibody detection and anti-SARS-CoV-2 specific monoclonal antibodies(m Abs) in the treatment of coronavirus infectious disease 2019(COVID-19). The dynamic changes of SARS-CoV-2 specific antibodies during COVID-19 were studied. Immunoglobulin M(Ig M) appeared earlier and lasted for a short time, while immunoglobulin G(Ig G) appeared later and last...     

Antibody response to third and fourth BNT162b2 mRNA booster vaccinations in healthcare workers in Tokyo,Japan

BackgroundBooster vaccinations against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are being promoted worldwide to counter the coronavirus disease 2019 (COVID-19) pandemic. In this study, we analyzed the longitudinal effect of the third BNT162b2 mRNA vaccination on antibody responses in healthcare workers. Additionally, antibody responses induced by the fourth vaccination were analyzed.MethodsThe levels of anti-spike (S) IgG and neutralizing antibody against SARS-CoV-2 were measured at 7 months after the second vaccination (n = 1138), and at 4 (n = 701) and 7 (n = 417) months after the third vaccination using an iFlash 3000 chemiluminescence immunoassay analyzer. Among the 417 participants surveyed at 7 months after the third vaccination, 40 had received the fourth vaccination. A multiple linear regression analysis was performed to clarify which factors were associated with the anti-S IgG and neutralizing antibody. Variables assessed included sex, age, number of days after the second or third vaccination, diagnostic history of COVID-19, and anti-nucleocapsid (N) IgG level.ResultsAt 7 months after the third vaccination, antibody responses were significantly higher than those at the same time after the second vaccination. Unlike the second vaccination, age had no effect on the antibody responses induced by the third vaccination. Furthermore, the fourth vaccination resulted in a further increase in antibody responses. The multiple linear regression analysis identified anti-N IgG level, presumably associated with infection, as a factor associated with antibody responses.ConclusionsOur findings showed that BNT162b2 booster vaccinations increased and sustained the antibody responses against SARS-CoV-2.     

SARS-CoV-2–specific antibody rearrangements in prepandemic immune repertoires of risk cohorts and patients with COVID-19

A considerable fraction of B cells recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with germline-encoded elements of their B cell receptor, resulting in the production of neutralizing and nonneutralizing antibodies. We found that antibody sequences from different discovery cohorts shared biochemical properties and could be retrieved across validation cohorts, confirming the stereotyped character of this naive response in coronavirus disease 2019 (COVID-19). While neutralizing antibody sequences were found independently of disease severity, in line with serological data, individual nonneutralizing antibody sequences were associated with fatal clinical courses, suggesting detrimental effects of these antibodies. We mined 200 immune repertoires from healthy individuals and 500 repertoires from patients with blood or solid cancers — all acquired prior to the pandemic — for SARS-CoV-2 antibody sequences. While the largely unmutated B cell rearrangements occurred in a substantial fraction of immune repertoires from young and healthy individuals, these sequences were less likely to be found in individuals over 60 years of age and in those with cancer. This reflects B cell repertoire restriction in aging and cancer, and may to a certain extent explain the different clinical courses of COVID-19 observed in these risk groups. Future studies will have to address if this stereotyped B cell response to SARS-CoV-2 emerging from unmutated antibody rearrangements will create long-lived memory.     

Anti-severe acute respiratory syndrome coronavirus-2 antibody responses following Pfizer-BioNTech vaccination in a patient with multiple sclerosis treated with ocrelizumab: a case report

Patients with multiple sclerosis (MS) repeatedly receive therapies that cause B-lymphocyte depletion. This may lead to abnormal immune responses following coronavirus disease 2019 (COVID-19) vaccination, as has been suggested previously. We therefore evaluated post-vaccination immune responses in a patient with MS treated with ocrelizumab. The intervals between ocrelizumab infusions and vaccination were as recommended by the Section of Multiple Sclerosis and Neuroimmunology of the Polish Neurological Society. A reactive immune response was observed in this patient following vaccination. This suggests that appropriate intervals between ocrelizumab infusions and COVID-19 vaccinations may permit the generation of efficacious immune responses in patients receiving B-lymphocyte depleting therapies.     

Transient immune hepatitis as post-coronavirus disease complication: A case report

BACKGROUNDWe report a case of post-coronavirus disease (COVID) immune hepatitis occurring in a young male with no pre-existing comorbidities.CASE SUMMARYA previously healthy 21-year-old male patient was admitted to our hospital with mild COVID-19. During the course of in-hospital isolation and monitoring, he developed an alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increase, with the enzymes peaking at day 24 (ALT 15 times the upper normal limit), with preserved liver function. The liver enzyme increase occurred 20 d after the complete clinical remission of COVID-19, and ALT dynamics paralleled the increase in total antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The case was interpreted as post-COVID immune hepatitis, with extensive laboratory investigations excluding other potential causes. The hepatocytolysis remitted 20 d after the peak ALT, without further intervention, with complete recovery, but the total anti-SARS-CoV-2 antibodies continued to increase the next 5 mo following the acute infection.CONCLUSIONClose attention should also be paid to young patients with mild forms of disease, and a high index of suspicion should be maintained for post-COVID complications.     

冻干无佐剂Vero细胞狂犬病疫苗免疫应答动态观察

目的评价国产冻干无佐剂Vero细胞狂犬病疫苗的免疫原性。方法选择既往无明确狂犬病疫苗接种史和犬伤史、符合研究方案制定的入选标准和排除标准为研究对象,对暴露于狂犬病的患者采用常规5针注射。观察对象于首针接种前、首针接种后7、14、28、45 d,全程后6个月采集血样检测抗狂犬病中和抗体。结果符合入选标准和排除标准的观察对象90名常规接种5针冻干无佐剂Vero细胞狂犬病疫苗。观察对象接种前狂犬病抗体均为阴性,接种首针后7 d狂犬病抗体阳转率为12.22%,接种首针后14 d阳转率达到100%,接种首针后28、45 d和全程后6个月的阳性率均为100%。接种首针后7 d抗狂犬病病毒综合抗体的几何抗体平均滴度（GMT）仅为0.27 IU/ml,接种首针后14 d狂犬病抗体的GMT达到2.52 IU/ml,较首针接种后7 d增长9.33倍。接种首针后28、45 d狂犬病抗体的GMT分别达到4.43、7.08 IU/ml,较首针后14 d分别增长1.76倍、2.81倍。全程接种后6个月狂犬病抗体的GMT仍达到8.41 IU/ml。结论国产冻干无佐剂Vero细胞狂犬病疫苗具有良好的免疫原性,6个月内再被暴露于狂犬病动物者可以不需要接种狂犬病疫苗。     

Asymptomatic COVID-19 re-infection in a Japanese male by elevated half-maximal inhibitory concentration (IC50) of neutralizing antibodies

Introduction“Re-infection” with COVID-19 is a growing concern; re-infection cases have reported worldwide. However, the clinical characteristics of SARS-CoV-2 re-infection, including the levels and role of anti-SARS-CoV-2 Spike protein IgG antibodies and the half-maximal concentration (IC₅₀) of neutralizing antibodies remain unknown.MethodsBoth the epidemiological and clinical information has been collected during two episodes of COVID-19 in a patient. Laboratory results, including RT-PCR, Ct values, anti-SARS-CoV-2 Spike protein IgG antibodies, and the IC₅₀ of neutralizing antibodies levels were analyzed on the patient.ResultsThe patient was a 58-year-old man who developed moderate COVID-19 pneumonia with oxygen demand (cannula 2 L/min) in the first episode. By day 30, he recuperated and was discharged after testing negative for SARS-CoV-2. After two and a half months, his three family members showed COVID-19 symptoms and tested positive for SARS-CoV-2. He tested positive for SARS-CoV-2 once again and was asymptomatic (the second episode). The IC₅₀ of neutralizing antibodies against SARS-CoV-2 greatly increased from 50.0 μg/mL (after the first episode) to 14.8 μg/mL (after the second episode), and remained strongly reactive (20.1 μl/mL) after 47 days of the second episode.ConclusionsEpidemiological, clinical, and serological analyses confirmed that the patient had re-infection instead of persistent viral shedding from first infection. Our results suggest that SARS-CoV-2 re-infection may manifest as asymptomatic with increased neutralizing antibody levels. Further studies such as the virus characteristics, immunology, and epidemiology on SARS-CoV-2 re-infection are needed.     

Review of an inactivated vaccine against hantaviruses

OBJECTIVE: Hantaviruses cause haemorrhagic fever with renal syndrome and result in severe morbidity and mortality in humans. Safe and effective vaccines are needed to reduce the incidence of human illness. In this study, the immune response to an inactivated hantavirus vaccine was measured in 64 human volunteers for Hantavax and 10 human volunteers for a Hantaan-Puumala virus combination vaccine at high risk of infection by virtue of their residence and occupation. METHODS: A serum sample was obtained from each volunteer before the initial vaccination (day 0), 30 days after each inoculation and 1 year after the initial dose. All sera were kept at -20 degrees until tested. IgG-specific antibody titres were tested by ELISA and immunofluorescence assay (IFA). Neutralizing antibody titres were determined by a plaque reduction neutralizing test. RESULTS: Thirty days after vaccination, 79 and 62% of the subjects had developed a significant hantavirus antibody titre as measured by IFA and ELISA, respectively. Seroconversion rates increased to 97% 1 month after the booster dose. Neutralizing antibody titres paralleled this trend, with 13% of vaccine recipients producing neutralizing antibody 1 month after the first dose and 75% of vaccine recipients responding 1 month after boosting. Antibody titres had declined by 1 year, however, with only 37 and 43% of sera found to be positive by IFA and ELISA, respectively. Re-vaccination at this time produced a vigorous anamnestic response, with 94 and 100% of vaccine recipients yielding positive antibody titres. Only 50% of the sampled population, however, produced neutralizing antibodies following the booster dose 1 year later. CONCLUSIONS: The vaccine was well tolerated and there were no apparent differences in the responses in human subjects. However, further improvement of this vaccine is necessary in order to induce a longer-lasting humoral immune response.     

Effectiveness of the third dose of BNT162b2 vaccine on neutralizing Omicron variant in the Japanese population

IntroductionThe vaccine against SARS-CoV-2 provides humoral immunity to fight COVID-19; however, the acquired immunity gradually declines. Booster vaccination restores reduced humoral immunity; however, its effect on newly emerging variants, such as the Omicron variant, is a concern. As the waves of COVID-19 cases and vaccine programs differ between countries, it is necessary to know the domestic effect of the booster.MethodsSerum samples were obtained from healthcare workers (20–69 years old) in the Pfizer BNT162b2 vaccine program at the Toyama University Hospital 6 months after the second dose (6mA2D, n = 648) and 2 weeks after the third dose (2wA3D, n = 565). The anti-SARS-CoV-2 antibody level was measured, and neutralization against the wild-type and variants (Delta and Omicron) was evaluated using pseudotyped viruses. Data on booster-related events were collected using questionnaires.ResultsThe median anti-SARS-CoV-2 antibody was >30.9-fold elevated after the booster (6mA2D, 710.0 U/mL [interquartile range (IQR): 443.0–1068.0 U/mL]; 2wA3D, 21927 U/mL [IQR: 15321.0–>25000.0 U/mL]). Median neutralizing activity using 100-fold sera against wild-type-, Delta-, and Omicron-derived variants was elevated from 84.6%, 36.2%, and 31.2% at 6mA2D to >99.9%, 99.1%, and 94.6% at 2wA3D, respectively. The anti-SARS-CoV-2 antibody levels were significantly elevated in individuals with fever ≥37.5 °C, general fatigue, and myalgia, local swelling, and local hardness.ConclusionThe booster effect, especially against the Omicron variant, was observed in the Japanese population. These findings contribute to the precise understanding of the efficacy and side effects of the booster and the promotion of vaccine campaigns.     

Analytical and clinical performance of cPass neutralizing antibodies assay

Serological tests for SARS-CoV-2 are a critical component of disease control strategies. SARS-CoV-2 serology tests used in clinical diagnostic should not accurately evaluate total levels the antibodies but also closely correlate with neutralizing antibodies titers.However, only limited data is available reporting correlation of neutralization antibody assays with commercial high-throughput serological assays widely used in clinical laboratories.We performed evaluation of the GenScript cPass neutralizing antibody detection assay, to assess its value for routine clinical use to measure neutralizing titers in patients who recovered from coronavirus disease 2019 (COVID-19) or have been vaccinated. We tested its clinical performance against the commonly used Ortho Vitros IgG assay.Our combined data shows that GenScript cPass neutralizing antibody assay has satisfactory analytical and clinical performance and good correlation with Ortho Vitros IgG, supporting its use as a tool for accurate SARS-COV-2 immune surveillance of recovered or vaccinated individuals.     

A case report of breakthrough infection with the SARS-CoV-2 delta variant and household transmission: Role of vaccination,anti-spike IgG and neutralizing activity

There have been several reports of breakthrough infections, which are defined as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections among individuals who had received at least two doses of vaccine at least 14 days before the onset of infection, but data on the antibody titers, including SARS-CoV-2 neutralizing antibody activity, and the clinical course of individuals with breakthrough infections are limited. We encountered a case of breakthrough infection with the SARS-CoV-2 delta variant in a 31-year-old female healthcare worker (the index case, Case 1) and a secondary case (Case 2) in her unvaccinated 33-year-old husband. We studied the role of the anti-spike immunoglobulin G (IgG) and neutralizing antibody activity in the two case patients. Case 1 had high anti-spike IgG detected on day 3 of the illness, with low neutralizing antibody activity. The neutralizing antibody activity started to increase on day 5 of the illness. In Case 2 both the anti-spike IgG and the neutralizing antibody activity remained low from days 4–11 of illness, and the anti-spike IgG gradually increased from day 9. In Case 1, the fever broke within 4 days of onset, coinciding with the rise in neutralizing antibodies, whereas the fever took 7 days to resolve in Case 2. SARS-CoV-2 infection can occur even in vaccinated individuals, but vaccination may contribute to milder clinical symptoms because neutralizing antibodies are induced earlier in vaccinated individuals than in unvaccinated individuals.     

Durable SARS-CoV-2 B cell immunity after mild or severe disease

Multiple studies have shown loss of severe acute respiratory syndrome coronavirus 2–specific (SARS-CoV-2–specific) antibodies over time after infection, raising concern that humoral immunity against the virus is not durable. If immunity wanes quickly, millions of people may be at risk for reinfection after recovery from coronavirus disease 2019 (COVID-19). However, memory B cells (MBCs) could provide durable humoral immunity even if serum neutralizing antibody titers decline. We performed multidimensional flow cytometric analysis of S protein receptor binding domain–specific (S-RBD–specific) MBCs in cohorts of ambulatory patients with COVID-19 with mild disease (n = 7), and hospitalized patients with moderate to severe disease (n = 7), at a median of 54 days (range, 39–104 days) after symptom onset. We detected S-RBD–specific class-switched MBCs in 13 of 14 participants, failing only in the individual with the lowest plasma levels of anti–S-RBD IgG and neutralizing antibodies. Resting MBCs (rMBCs) made up the largest proportion of S-RBD–specific MBCs in both cohorts. FCRL5, a marker of functional memory on rMBCs, was more dramatically upregulated on S-RBD–specific rMBCs after mild infection than after severe infection. These data indicate that most SARS-CoV-2-infected individuals develop S-RBD–specific, class-switched rMBCs that resemble germinal center–derived B cells induced by effective vaccination against other pathogens, providing evidence for durable B cell–mediated immunity against SARS-CoV-2 after mild or severe disease.     

First-dose mRNA vaccination is sufficient to reactivate immunological memory to SARS-CoV-2 in subjects who have recovered from COVID-19

The characterization of the adaptive immune response to COVID-19 vaccination in individuals who recovered from SARS-CoV-2 infection may define current and future clinical practice. To determine the effect of the 2-dose BNT162b2 mRNA COVID-19 vaccination schedule in individuals who recovered from COVID-19 (COVID-19–recovered subjects) compared with naive subjects, we evaluated SARS-CoV-2 Spike–specific T and B cell responses, as well as specific IgA, IgG, IgM, and neutralizing antibodies titers in 22 individuals who received the BNT162b2 mRNA COVID-19 vaccine, 11 of whom had a previous history of SARS-CoV-2 infection. Evaluations were performed before vaccination and then weekly until 7 days after second injection. Data obtained clearly showed that one vaccine dose is sufficient to increase both cellular and humoral immune response in COVID-19–recovered subjects without any additional improvement after the second dose. On the contrary, the second dose proved mandatory in naive subjects to further enhance the immune response. These findings were further confirmed at the serological level in a larger cohort of naive (n = 68) and COVID-19–recovered (n = 29) subjects, tested up to 50 days after vaccination. These results question whether a second vaccine injection in COVID-19–recovered subjects is required, and indicate that millions of vaccine doses may be redirected to naive individuals, thus shortening the time to reach herd immunity.     

Efficient immunization of rhesus macaques with an HCV candidate vaccine by heterologous priming-boosting with novel adenoviral vectors based on different serotypes

Efficient vaccination against viral agents requires a strong T-cell-mediated immune response to clear viral-infected cells. Optimal vaccination can be achieved by administration of recombinant viral vectors encoding phatogen antigens. Adenoviral vectors have attracted considerable attention as potential viral vectors for genetic vaccination owing to their favorable safety profile and potent transduction efficiency following intramuscular injection. However, the neutralizing antibody response against adenoviral capsid proteins following adenoviral vectors injection limits the success of vaccination protocols based on multiple administrations of the same adenoviral serotype. In this work, we describe efficient immunization of rhesus macaques, the preferred model for preclinical assessment, with an HCV candidate vaccine by heterologous priming-boosting with adenoviral vectors based on different serotypes. The induced responses are broad and show significant cross-strain reactivity. Boosting can be delayed for over 2 years after priming, indicating that there is long-term maintenance of resting memory cells.     

Donors for SARS-CoV-2 Convalescent Plasma for a Controlled Clinical Trial: Donor Characteristics,Content and Time Course of SARS-CoV-2 Neutralizing Antibodies

BackgroundConvalescent plasma is one of the treatment options for COVID-19 which is currently being investigated in many clinical trials. Understanding of donor and product characteristics is important for optimization of convalescent plasma.MethodsPatients who had recovered from CO­VID-19 were recruited as donors for COVID-19 convalescent plasma (CCP) for a randomized clinical trial of CCP for treatment of severe COVID-19 (CAPSID Trial). Titers of neutralizing antibodies were measured by a plaque-reduction neutralization test (PRNT). Correlation of antibody titers with host factors and evolution of neutralizing antibody titers over time in repeat donors were analysed.ResultsA series of 144 donors (41% females, 59% males; median age 40 years) underwent 319 plasmapheresis procedures providing a median collection volume of 850 mL and a mean number of 2.7 therapeutic units per plasmapheresis. The majority of donors had a mild or moderate course of COVID-19. The titers of neutralizing antibodies varied greatly between CCP donors (from <1:20 to >1:640). Donor factors (gender, age, ABO type, body weight) did not correlate significantly with the titer of neutralizing antibodies. We observed a significant positive correlation of neutralization titers with the number of reported COVID-19 symptoms and with the time from SARS-CoV-2 diagnosis to plasmapheresis. Neutralizing antibody levels were stable or increased over time in 58% of repeat CCP donors. Mean titers of neutralizing antibodies of first donation and last donation of repeat CCP donors did not differ significantly (1:86 at first compared to 1:87 at the last donation). There was a significant correlation of neutralizing antibodies measured by PRNT and anti-SARS-CoV-2 IgG and IgA antibodies which were measured by ELISA. CCP donations with an anti-SARS-CoV-2 IgG antibody content above the 25th percentile were substantially enriched for CCP donations with higher neutralizing antibody levels.ConclusionWe demonstrate the feasibility of collection of a large number of CCP products under a harmonized protocol for a randomized clinical trial. Titers of neutralizing antibodies were stable or increased over time in a subgroup of repeat donors. A history of higher number of COVID-19 symptoms and higher levels of anti-SARS-CoV-2 IgG and IgA antibodies in immunoassays can preselect donations with higher neutralizing capacity.     

Potent immune response against HIV-1 and protection from virus challenge in hu-PBL-SCID mice immunized with inactivated virus-pulsed dendritic cells generated in the presence of IFN-alpha

A major challenge of AIDS research is the development of therapeutic vaccine strategies capable of inducing the humoral and cellular arms of the immune responses against HIV-1. In this work, we evaluated the capability of DCs pulsed with aldrithiol-2-inactivated HIV-1 in inducing a protective antiviral human immune response in SCID mice reconstituted with human PBL (hu-PBL-SCID mice). Immunization of hu-PBL-SCID mice with DCs generated after exposure of monocytes to GM-CSF/IFN-alpha (IFN-DCs) and pulsed with inactivated HIV-1 resulted in a marked induction of human anti-HIV-1 antibodies, which was associated with the detection of anti-HIV neutralizing activity in the serum. This vaccination schedule also promoted the generation of a human CD8+ T cell response against HIV-1, as measured by IFN-gamma Elispot analysis. Notably, when the hu-PBL-SCID mice immunized with antigen-pulsed IFN-DCs were infected with HIV-1, inhibition of virus infection was observed as compared with control animals. These results suggest that IFN-DCs pulsed with inactivated HIV-1 can represent a valuable approach of immune intervention in HIV-1-infected patients.     

A Novel Replication-Competent Vaccinia Vector MVTT Is Superior to MVA for Inducing High Levels of Neutralizing Antibody via Mucosal Vaccination

Mucosal vaccination offers great advantage for inducing protective immune response to prevent viral transmission and dissemination. Here, we report our findings of a head-to-head comparison of two viral vectors modified vaccinia Ankara (MVA) and a novel replication-competent modified vaccinia Tian Tan (MVTT) for inducing neutralizing antibodies (Nabs) via intramuscular and mucosal vaccinations in mice. MVTT is an attenuated variant of the wild-type VTT, which was historically used as a smallpox vaccine for millions of Chinese people. The spike glycoprotein (S) of SARS-CoV was used as the test antigen after the S gene was constructed in the identical genomic location of two vectors to generate vaccine candidates MVTT-S and MVA-S. Using identical doses, MVTT-S induced lower levels (~2-3-fold) of anti- SARS-CoV neutralizing antibodies (Nabs) than MVA-S through intramuscular inoculation. MVTT-S, however, was capable of inducing consistently 20-to-100-fold higher levels of Nabs than MVA-S when inoculated via either intranasal or intraoral routes. These levels of MVTT-S-induced Nab responses were substantially (~10-fold) higher than that induced via the intramuscular route in the same experiments. Moreover, pre-exposure to the wild-type VTT via intranasal or intraoral route impaired the Nab response via the same routes of MVTT-S vaccination probably due to the pre-existing anti-VTT Nab response. The efficacy of intranasal or intraoral vaccination, however, was still 20-to-50-fold better than intramuscular inoculation despite the subcutaneous pre-exposure to wild-type VTT. Our data have implications for people who maintain low levels of anti-VTT Nabs after historical smallpox vaccination. MVTT is therefore an attractive live viral vector for mucosal vaccination.     

Studies on the in vitro formation of antibodies. III. Induction of primary and secondary immune response in vitro]

Peritoneal cells from normal, unimmunized mice (female NMRI, 28-32 gr) produced in vitro primary and secondary immune response after induction with the bacteriophage T2 6 hours or 7 day resp. after establishing the cultures. We confirmed the induction of a primary and secondary immunological response in vitro in the very same culture by the following data: 1. In vivo the donor animals were not in contact with the antigen used. We found neither the phage nor its host E. coli B in the gut of 97 mice investigated and no humoral antibodies against T2. The kinetics of humoral antibody production in vivo by different doses of T2 also showed that there are no related or identical antigen structures incorporated in our animals. 2. The T2 neutralizing activity in the culture medium after the first induction had the sedimentation constant of 19.7 +/- 2.3 S (n = 9) but the activity found after the second induction sedimented with 8.1 +/- 0.7 S (n = 10). 3. The primary activity was more sensitive to mercaptoethanol than the secondary. 4. Complement was bound by the complex T2 + neutralizing activity.     

Single-dose replicating poxvirus vector-based RBD vaccine drives robust humoral and T cell immune response against SARS-CoV-2 infection

The coronavirus disease 2019 (COVID-19) pandemic requires the continued development of safe, long-lasting, and efficacious vaccines for preventive responses to major outbreaks around the world, and especially in isolated and developing countries. To combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we characterize a temperature-stable vaccine candidate (TOH-Vac1) that uses a replication-competent, attenuated vaccinia virus as a vector to express a membrane-tethered spike receptor binding domain (RBD) antigen. We evaluate the effects of dose escalation and administration routes on vaccine safety, efficacy, and immunogenicity in animal models. Our vaccine induces high levels of SARS-CoV-2 neutralizing antibodies and favorable T cell responses, while maintaining an optimal safety profile in mice and cynomolgus macaques. We demonstrate robust immune responses and protective immunity against SARS-CoV-2 variants after only a single dose. Together, these findings support further development of our novel and versatile vaccine platform as an alternative or complementary approach to current vaccines.