कोरोना भाइरस विरुद्धको भ्याक्सिन बनाउँदा चीनले पायो ठूलो सफलता (पूर्ण प्रतिवेदनसहित)

Background

Methods

Findings

Interpretation

Funding

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in January, 2020. The virus is highly transmissible between humans and has spread rapidly, causing the COVID-19 pandemic. ^,    Patients infected with SARS-CoV-2, especially older patients and those with pre-existing respiratory or cardiovascular conditions are at greater risk for severe complications, including severe pneumonia, acute respiratory distress syndrome, multiple organ failure, and in some cases, death. ^,    By May 20, 2020, SARS-CoV-2 had infected more than 4·7 million people across 215 countries or territories and killed more than 316 000 worldwide.

In the absence of effective prevention measures, current management to control the epidemic is the enforcement of quarantine, isolation, and physical distancing. ^,    Effective vaccines against COVID-19 are urgently needed to reduce the enormous burden of mortality and morbidity associated with SARS-CoV-2 infection.  There are more than 100 candidate vaccines in development worldwide,  among them at least eight have started or will soon start clinical trials. These include Moderna's mRNA COVID-19 vaccine and CanSino's non-replicating adenovirus type-5 (Ad5) vectored COVID-19 vaccine, which both entered phase 1 clinical trials on March 16, 2020; Inovio Pharmaceuticals' DNA vaccine for COVID-19, which entered trials on April 3, 2020; three inactive COVID-19 vaccines manufactured by Sinovac, Wuhan Institute of Biological Products, and Beijing Institute of Biological Products entered clinical trials in April, 2020, successively; University of Oxford's non-replicating chimpanzee adenovirus vectored vaccine ChAdOx1 nCoV-19, and BioNTech's mRNA COVID-19 vaccine also started trials in recent months.

Here, we report the preliminary assessment at 28 days post-vaccination of the safety, tolerability, and immunogenicity of CanSino's non-replicating Ad5 vectored COVID-19 vaccine in healthy adults in China.

Between March 16 and March 27, 2020, we screened 195 individuals for eligibility. Of them, 108 were sequentially enrolled and assigned to receive the low dose (n=36 [33%]), middle dose (n=36 [33%]), or high dose (n=36 [33%]) of the Ad5 vectored COVID-19 vaccine (appendix p 2). All participants completed the vaccination and the scheduled visits within 28 days. Baseline characteristics of the participants were similar across the treatment groups (table 1).

87 (81%) of 108 participants reported at least one adverse reaction within the first 7 days after the vaccination: 30 (83%) in the low dose group, 30 (83%) in the middle dose group, and 27 (75%) in the high dose group (table 2). No significant difference in the overall number of adverse reactions across the treatment groups was observed. The most common injection site adverse reaction was pain, which was reported in 58 (54%) vaccine recipients. Pain was reported in 17 (47%) participants in the low dose group, 20 (56%) participants in the middle dose group, and 21 (58%) participants in the high dose group. The most commonly reported systematic adverse reactions overall were fever (50 [46%]), fatigue (47 [44%]), headache (42 [39%]), and muscle pain (18 [17%]). Fever was reported in 15 (42%) participants in the low dose group, 15 (42%) participants in the middle dose group, and 20 (56%) participants in the high dose group. Headache was reported in 14 (39%) participants in the low dose group, 11 (31%) participants in the middle dose group, and 17 (47%) participants in the high dose group. Muscle pain was reported in seven (19%) participants in the low dose group, three (8%) participants in the middle dose group, and eight (22%) participants in the high dose group. Most adverse reactions were mild or moderate in severity. Nine participants (two [6%] in the low dose group, two [6%] in the middle dose group, and five [14%] in the high dose group) had an episode of severe fever (grade 3) with axillary temperature greater than 38·5°C. Of them, one (3%) from the high dose group reported severe fever along with severe symptoms of fatigue, dyspnoea, and muscle pain. One participant in the high dose group reported severe fatigue and joint pain (appendix p 3). These reactions occurred within 24 h post-vaccination, and persisted for no more than 48 h. We found no significant difference in the incidences of adverse reactions or overall adverse events among the dose groups. High pre-existing Ad5 immunity (titre of >1:200 vs ≤1:200) was associated with significantly fewer occurrences of fever post-vaccination (odds ratio 0·3, 95% CI 0·1–0·6; appendix p 4). No serious adverse event was reported within 28 days. At day 7 after vaccination, nine (8%) participants had mild to moderate total bilirubin increase, ten (9%) had alanine aminotransferase increase, and four (4%) had fasting hyperglycaemia (appendix p 5), but no instances were considered as clinically significant.

Rapid binding antibody responses to RBD were observed in all three dose groups from day 14 (table 3). At day 28, the recipients in the high dose group tended to have a higher binding antibody geometric mean titre of 1445·8 (95% CI 935·5–2234·5), followed by 806·0 (528·2–1229·9) in the middle dose group, and 615·8 (405·4–935·5) in the low dose group (high dose vs low dose 1611·5, 531·5–2691·5). At least a four-fold increase of anti-RBD antibodies was noted in 35 (97%) of 36 participants in the low dose group, 34 (94%) of 36 in the middle dose group, and 36 (100%) of 36 in the high dose group. Neutralising antibodies against live SARS-CoV-2 were all negative at day 0, and increased moderately at day 14, peaking at 28 days post-vaccination. Neutralising antibody titre with a geometric mean titre of 34·0 (95% CI 22·6–50·1) was noted in the high dose group, which was significantly higher compared with 16·2 (10·4–25·2) in the middle dose group and 14·5 (9·6–21·8) in the low dose group, with an estimated difference of 27·7 (1·0–54·4) between the high dose group and the middle dose group and 33·2 (6·5–59·9) between the high dose group and the low dose group at day 28. Meanwhile, 18 (50%) participants in the low dose group, 18 (50%) in the middle dose group, and 27 (75%) in the high dose group had at least a four-fold increase in neutralising antibody titres by day 28. Similar patterns of the binding antibody to spike glycoprotein and neutralising antibody titre to pseudovirus post-vaccination across the dose groups were also noted (appendix p 6). The association between the ELISA antibodies to RBD and neutralising antibody titres against live virus showed a moderate positive correlation of 0·749, and that between the ELISA antibodies to spike glycoprotein and neutralising antibody titres against live virus was 0·753, at the peak antibody response (p<0·0001). The neutralising antibody titres measured using a pseudovirus were also correlated well with those measured by live SARS-CoV-2 (appendix p 7).

Before vaccination, 20 (56%) participants in the low dose group, 19 (53%) participants in the middle dose group, and 16 (44%) participants in the high dose group had a high pre-existing Ad5 neutralising antibody titre (>1:200). Only five (25%) participants of 20 in the low dose group, seven (37%) participants of 19 in the middle dose group, and ten (63%) participants of 16 in the high dose group, who had high pre-existing Ad5 immunity, had at least a four-fold increase in neutralising antibody titre at day 28 post-vaccination (appendix pp 8–10). Multivariable analysis showed that high pre-existing Ad5 neutralising antibody titres compromised the seroconversion of neutralising antibody post-vaccination, regardless of the vaccine doses, and recipients aged 45–60 years seemed to have lower seroconversion of neutralising antibody compared with the younger recipients (appendix p 11). The Ad5 neutralising antibodies were significantly boosted post-vaccination (appendix p 12).

ELISpot responses at baseline were undetectable with spot-forming cells below the level of detection of the assay in all participants, but peaked at day 14 post-vaccination. The proportions of positive responders ranged from 83–97% across the dose groups, with a mean number of spot-forming cells per 100 000 cells of 20·8 (95% CI 12·7–34·0) in the low dose group, 40·8 (27·6–60·3) in the middle dose group, and 58·0 (39·1–85·9) in the high dose group (figure 1). T-cell responses in the high dose group were significantly higher than that in the low dose group (p<0·0010), but not significant compared with that in the middle dose group. A slight decrease of the T-cell responses across the dose groups was noted at day 28. High levels of baseline Ad5 neutralising antibody titre reduced the peak of post-vaccination T-cell responses in all the dose groups, particularly for the low dose group. Despite the effect of high pre-existing Ad5 immunity, positive responders were identified in 15 (75%) of 20 participants in the low dose group, 18 (95%) of 19 participants in the middle dose group, and 15 (94%) of 16 participants in the high dose group at day 14, and 12 (60%) of 20 participants in the low dose group, 16 (84%) of 19 participants in the middle dose group, and 16 (100%) of 16 participants in the high dose group at day 28.

IFNγ was detected from CD4⁺ and CD8⁺ T cells after the vaccination at day 14 and 28, in all dose groups (figure 2, appendix p 13). The TNFα expression from CD4⁺ T cells tended to be significantly lower in the low dose group than that in the high dose (p<0·0001) and middle dose groups (p=0·0032), on day 14. The TNFα expression from CD8⁺ T cells showed an overall p value of less than 0·0001 across the three groups on day 14. And the TNFα expression from CD8⁺ T cells tended to be higher in the high dose group than that in both the middle dose group (p=0·016) and the low dose group (p<0·0001). The p values are for the pairwise comparisons between groups. Amounts of IL-2 detected from CD4⁺ T cells were higher than that detected from CD8⁺ cells. The proportions of polyfunctional phenotypes detected from memory CD4⁺ T cells were higher than those from CD8⁺ T cells. Higher proportions of polyfunctional phenotypes were noted with the higher vaccine doses. We also noted that pre-existing Ad5 neutralising antibody had a negative effect on the pattern of T-cell responses (appendix pp 14–16). A post-hoc analysis showed that 28 (78%) participants in the low dose group, 33 (92%) participants in the middle dose group, and 36 (100%) participants in the high dose group showed either positive T-cell responses to spike glycoprotein or seroconversion of neutralising antibody to live SARS-CoV-2, at day 28 post-vaccination (appendix p 17).

To exclude any possible SARS-CoV-2 exposure during the study period, we tested the serum antibodies to nucleocapsid protein of SARS-CoV-2 in participants at day 28 using a special IgG/IgM rapid test kit (Vazyme Biotech, number CD101, Nanjing, China), but none of the participants were positive.

To our knowledge, this is the first report on a first-in-human clinical trial of a novel Ad5 vectored COVID-19 vaccine. The Ad5 vectored COVID-19 vaccine was tolerated in healthy adults in all three dose groups. The most common adverse reactions were fever, fatigue, headache, and muscle pain with no significant difference in the incidence of adverse reactions across the groups. Most adverse events reported were mild or moderate in severity. We noticed a higher reactogenicity profile of the high dose at 1·5 × 10¹¹ viral particles, presenting as severe fever, fatigue, muscle pain, or joint pain, which might be associated with viraemia caused by Ad5 vector infection. However, the severe adverse reactions were transient and self-limiting. Additionally, no abnormal changes in laboratory measurements were clinically significant or considered to be related to the vaccine. The profile of adverse events reported in this trial is similar to that of another Ad5 vector-based Ebola vaccine expressing glycoprotein.  To accelerate the process of clinical evaluation of the candidate COVID-19 vaccine, we selected doses for the phase 2 study mainly on the basis of the safety profile of the candidate vaccines shown in the participants within 7 days and 14 days post-vaccination. We chose the low dose (5 × 10¹⁰ viral particles) and middle dose (1 × 10¹¹ viral particles) to be further assessed in a phase 2 clinical trial.

The Ad5 vectored COVID-19 vaccine was immunogenic, inducing humoral and T-cell responses rapidly in most participants. Onset of detectable immune responses was rapid, with T-cell responses peaking at day 14 after vaccination and antibodies peaking at day 28. The antibody response to the vaccine in the high dose group was slightly greater than that in the middle dose and low dose groups. A single dose of Ad5 vectored COVID-19 vaccine was able to elicit a four-fold increase in binding antibodies to RBD in 94–100% of participants, and a four-fold increase to live virus in 50–75% of participants. Despite differences in magnitudes of the antibodies measured through different methods, there was a strong positive correlation between binding antibodies and neutralising antibody titres to the live virus. High proportions of participants with positive T-cell responses were noted across the all dose groups post-vaccination. The activation of both CD4⁺ T cells and CD8⁺ T cells was observed in vaccine recipients, particularly for antigen-specific CD4⁺ T cells and CD8⁺ T cells. However, both the specific antibody response and T-cell response induced by vaccination were partly diminished by the presence of high pre-existing anti-Ad5 immunity.

Currently, correlates of protection for a vaccine against COVID-19 are unknown, and the roles of the specific antibodies or T cells in building effective protection are not yet defined. Therefore, we are unable to predict the protection of the Ad5 vectored COVID-19 vaccine on the basis of the vaccine-elicited immune responses in this study. However, previous studies investigating SARS and Middle East respiratory syndrome (MERS) found that the increases in specific antibodies were temporary, and declined quickly in patients after recovery, whereas the specific CD4⁺ and CD8⁺ T-cell responses played an essential role in immunity. ^,    A similar rapid decline of the specific antibody amounts in patients with COVID-19 after recovery was also noted, ^,    suggesting that both specific cellular and humoral immunity are potentially important for a successful COVID-19 vaccine. Here, we only report the data within 28 days after the vaccination, but we are going to follow up the vaccine recipients for at least 6 months, so more data will be obtained.

This study was done in Wuhan, Hubei province, which was the centre of the COVID-19 epidemic in China.  People living in the city of Wuhan had a much higher risk of SARS-CoV-2 infection compared with those living in other cities outside of Hubei province, even though when we initiated this trial, the city had already begun lockdown and implemented mandatory home isolation for residents. Therefore, we did serological screening, nucleic acid testing, and chest CT to exclude participants who had been previously exposed to SARS-CoV-2 during recruitment. In addition, we arranged for all participants in our study to stay in a designated hotel for 14 days post-vaccination. This arrangement facilitated the observation of adverse events after the immunisation of the participants, and reduced the risk of SARS-CoV-2 exposure during the following 2 weeks. These measures allowed the study to be done successfully without interference by the circulation of SARS-CoV-2, which is especially important in the absence of a placebo control.

Interpretation of the results of this study is limited by the small size of the cohort, the short duration of follow-up, and the absence of a randomised control group. As it was a first-in-human study of the Ad5 vectored COVID-19 vaccine, it was not designed to measure the vaccine efficacy. However, in preclinical studies, seven out of eight ferrets were protected from having detectable virus copies when challenged by SARS-CoV-2 through nasal dripping 21 days after immunisation with the vaccine, whereas only one out of eight ferrets in the control group was negative for virus copies (Wei C, unpublished). We aimed to evaluate the safety and tolerability of the candidate vaccine in healthy adults, with no interference by underlying diseases or medicines. However, results of our study indicated that older age could have a negative effect on the vaccine-elicited responses to SARS-CoV-2. In this trial, no participants were older than 60 years and only 16% of the participants were older than 50 years, providing limited information on the capability of generating a potent cellular and humoral response in the older population. Since age has also been identified as an independent risk factor for severe disease associated with SARS-CoV-2 infection, ^,    and there is a possibility that an even lower immune response might be found in the older population, we are going to include participants who are older than 60 years in the phase 2 study considering this population as an important target population for a COVID-19 vaccine. Additionally, experience with vaccine candidates for SARS and MERS have raised concerns about the antibody-dependent enhancement in participants who are infected with a circulating SARS-CoV-2 post-vaccination.  However, this study was not statistically powered to measure any safety outcome, especially for the concerns around immunopathology and antibody-dependent enhancement events associated with the full-length spike glycoprotein vaccine antigen.  Our study found that the pre-existing Ad5 immunity could slow down the rapid immune responses to SARS-CoV-2 and also lower the peak of the responses, particularly for humoral immunity. The high pre-existing Ad5 immunity might also have a negative effect on the persistence of the vaccine-elicited immune responses. In previous studies, heterologous prime-boost combinations or homologous prime-boost regimens with Ad5 vectored vaccines were shown to be able to induce more strong and durable immunogenic responses in populations with high pre-existing Ad5 immunity. ^{,   ,}    Nevertheless, limited information is available for the effects of multiple doses of the candidate Ad5 vectored COVID-19 vaccine in humans, which warrants further investigation.

Over the past decade, the vaccine industry and clinical research centres have been asked to provide urgent responses to epidemics of emerging infectious diseases, such as H1N1 influenza, Ebola virus, Zika, MERS, and now SARS-CoV-2.  The risk of COVID-19 caused by SARS-CoV-2 is ongoing, making the need for effective vaccines even more urgent.  We started the development of this candidate vaccine in January, 2020, when SARS-CoV-2 was first isolated and sequenced. The full-length spike glycoprotein was selected as the vaccine antigen, mainly on the basis of previous experience with SARS and MERS vaccines. Previous findings suggested that those vaccines expressing full-length spike glycoprotein can induce good immune responses and protective efficacy.  Although the RBD comprises the critical neutralising domains for the coronaviruses, the neutralising epitopes located outside the RBD were also identified. ^,    The full-length spike was chosen in most of the viral vectored, mRNA, or DNA COVID-19 vaccines in development.  The Ad5 vector vaccine platform is highly efficient and well established as a vaccine antigen delivery system. In addition to our candidate Ad5 vectored COVID-19 vaccine, there are several other Ad5-based vaccines against COVID-19 listed in the WHO draft landscape of COVID-19 candidate vaccines, including Ad5 S (GREVAXTM platform) in the USA, and Oral Ad5 S (Stabilitech Biopharma) in the UK.  However, aside from pre-existing anti-Ad5 immunity, there is a concern about the increased risk of HIV-1 acquisition associated with Ad5 activated CD4⁺ T cells.  Although the association between HIV-1 acquisition risk and Ad5 vectored vaccine is controversial and its mechanism is unclear, the potential risks should be taken into account in studies with this viral vector delivery platform. We plan to monitor the participants in our upcoming phase 2 and phase 3 studies to assess the indication for any such acquisition.

In conclusion, we found that the Ad5 vectored COVID-19 vaccine is tolerable and immunogenic in healthy adults. Specific humoral responses against SARS-CoV-2 peaked at day 28 post-vaccination, and rapid, specific T-cell responses were noted from day 14 after one shot of the vaccine. There is potential for further investigation of the Ad5 vectored COVID-19 vaccine for the control of the COVID-19 outbreak. An ongoing phase 2 trial in China (NCT04341389) will provide more information on the safety and immunogenicity of the Ad5 vectored COVID-19 vaccine.

We support data sharing of the individual participant data. The individual participant data that underlie the results reported in this Article, after de-identification (text, tables, figures, and appendix) will be shared. Individual participant data will be available beginning 3 months and ending 1 year after publication. Supporting clinical documents including the study protocol, statistical analysis plan, and the informed consent form will be available immediately following publication for at least 1 year. Researchers who provide a scientifically sound proposal will be allowed access to the individual participant data. Proposals should be directed to [email protected] or [email protected]. These proposals will be reviewed and approved by the sponsor, investigator, and collaborators on the basis of scientific merit. To gain access, data requesters will need to sign a data access agreement.

We thank Peng Deng, Qiong Li, and Xiaoai Qian from Hubei Provincial Center for Disease Control and Prevention for participant recruitment and sample collection. We thank Miao Xu and Jingjing Liu from the National Institute for Food and Drug Control (China), Yansong Sun, Sen Zhang, and Yuchang Li from the Beijing Institute of Microbiology and Epidemiology, and Feng Wang, Hongyan Hou, Hanxiong Guan, and Bo Liu from Tongji Hospital for laboratory analysis. We thank Ke Zhang from the Academy of Military Medical Sciences, Kun Liu from the General Hospital of Central Theater Command, and Changlong Fu from Wuhan Rest Center, Chinese People's Armed Police Force for the management of the clinical trial site.