The vaccine also blocks animal-to-animal transmission of the virus. The findings were published July 2 in the journal Science Advances.
“The currently available vaccines against COVID-19 are very successful, but the majority of the world’s population is still unvaccinated and there is a critical need for more vaccines that are easy to use and effective at stopping disease and transmission,” says Paul McCray, MD, professor of pediatrics-pulmonary medicine, and microbiology and immunology at the UI Carver College of Medicine, and co-leader of the study.
Unlike traditional vaccines that require an injection, this vaccine is administered through a nasal spray similar to those commonly used to vaccinate against influenza. The vaccine used in the study only requires a single dose and it may be stored at normal refrigerator temperatures for up to at least three months.
Because it is given intranasally, the vaccine may also be easier to administer, especially for those who have a fear of needles.
“We have been developing this vaccine platform for more than 20 years, and we began working on new vaccine formulations to combat COVID-19 during the early days of the pandemic,” says Biao He, Ph.D., a professor in the University of Georgia’s Department of Infectious Diseases in the College of Veterinary Medicine and co-leader of the study.
“Our preclinical data show that this vaccine not only protects against infection, but also significantly reduces the chances of transmission.”
The research team has previously shown that this vaccine platform can completely protect experimental animals from another dangerous coronavirus disease called Middle Eastern Respiratory Syndrome (MERS).
The inhaled PIV5 vaccine developed by the team targets mucosal cells that line the nasal passages and airways. These cells are the main entry point for most SARS-CoV-2 infections and the site of early virus replication. Virus produced in these cells can invade deeper into the lungs and other organs in the body, which can lead to more severe disease. In addition, virus made in these cells can be easily shed through exhalation allowing transmission from one infected person to others.
The study showed that the vaccine produced a localized immune response, involving antibodies and cellular immunity, that completely protected mice from fatal doses of SARS-CoV-2.
The vaccine also prevented infection and disease in ferrets and, importantly, appeared to block transmission of COVID-19 from infected ferrets to their unprotected and uninfected cage-mates.
Middle East respiratory syndrome (MERS) emerged as a significant illness on the Saudi Arabian peninsula in mid-2012, and the causative agent was identified as a novel coronavirus (CoV), MERS-CoV (1). MERS has a high mortality rate (∼35%) associated with severe lung disease that can advance to acute respiratory distress syndrome (ARDS). MERS-CoV, similarly to SARS-CoV, which caused a similar epidemic in 2003, has been a global cause for concern due to its high fatality rate.
Epidemiologic studies established that MERS-CoV is zoonotic in origin, with transmission occurring from dromedary camels on the Arabian peninsula (2–4). Spread from camels to people is documented (5), as well as person-to-person spread among health care workers in hospital settings (6). To date, MERS-CoV has spread to 27 countries and caused 858 deaths in 2,494 confirmed cases (4 February 2020, World Health Organization [WHO]), including a large travel-related outbreak in South Korea in 2015 (7).
MERS-CoV is an enveloped positive-stranded RNA virus whose entry into target cells is mediated by the viral envelope S protein. The S protein consists of an S1 subunit responsible for binding to the virus receptor, dipeptidyl peptidase 4 (DPP4 or CD26), via a receptor-binding domain (RBD), and an S2 subunit that mediates membrane fusion (8–10). Thus, the S protein, particularly the RBD, is an important target for MERS-CoV vaccine development (8, 11, 12).
There is currently no vaccine or antiviral therapeutic against MERS-CoV. A number of candidate MERS-CoV vaccines, including those based on recombinant virus, viral vectors (e.g., MVA, adenovirus, and measles virus), nanoparticles, DNA, and DNA/protein, as well as subunit vaccines, are under development (12, 13). None are approved; thus, the need remains for an effective and broad-spectrum vaccine against MERS-CoV infection (14).
PIV5, formerly known as simian virus 5 (SV5), is a nonsegmented, negative-strand, RNA virus (NNSV). It is a member of the Rubulavirus genus of the family Paramyxoviridae, which includes mumps virus (MuV) and human parainfluenza virus type 2 (HPIV2) and type 4 (HPIV4) (15). PIV5 encodes eight known viral proteins (15).
Nucleocapsid protein (NP), phosphoprotein (P), and large RNA polymerase (L) protein are important for transcription and replication of the viral RNA genome. PIV5 is an excellent viral vector candidate for vaccine development; it is safe and infects a large number of mammals without being associated with any diseases, except kennel cough in dogs (16–20).
Because PIV5 does not have a DNA phase in its life cycle, its use avoids the possible unintended consequences of genetic modifications of host cell DNA through recombination or insertion. In comparison to positive-strand RNA viruses, the genome structure of PIV5 is stable. A recombinant PIV5 expressing F of respiratory syncytial virus (RSV) has been generated, and the F gene was maintained for more than 10 generations (21).
PIV5 can be grown to 8 × 108 PFU/ml, indicating its potential as a cost-effective and safe vaccine vector that may be used in mass production. We have discovered that PIV5-based influenza, respiratory syncytial virus (RSV), and rabies vaccines are efficacious (22–28).
In studies of influenza, we previously reported that that a PIV5 vector expressing influenza virus NA provided sterilizing immunity (no mortality, no morbidity, and no virus detected in the lungs of challenged mice at 4 days postchallenge) and PIV5 expressing NP protected 100% of mice against lethal influenza virus H1N1 challenge in mice (25), demonstrating that PIV5 is an excellent vector for developing vaccines for respiratory pathogens. Here we investigate the utility of a PIV5-based vaccine expressing the MERS S protein in a robust humanized mouse model of lethal MERS-CoV infection.
DISCUSSION
Many strategies have been considered to develop vaccines for both SARS-CoV and MERS-CoV. A live attenuated SARS-CoV with rationally introduced mutations was efficacious in golden Syrian hamsters (30). However, the development of a live attenuated vaccine for a positive-stranded RNA virus like SARS-CoV has often been hampered by safety concerns. Several MERS vaccine candidates are under investigation.
A DNA-based vaccine expressing the full-length S protein is the most advanced to date (31); it is well tolerated in humans, as shown in a phase I clinical trial. The prime-boost regimen of MVA (Modified Vaccinia Ankara) expressing MERS S protein induced neutralizing antibodies and T cell responses in mice and limited viral replication after challenge in mice and camels. However, MVA-S did not provide sterilizing immunity, and infectious MERS-CoV and genomic RNA were detected after challenge in mice and camels (32, 33).
The prime-boost regimen of measles virus (MV) expressing MERS S or soluble S induced both humoral and cellular immune responses. After MERS challenge, infectious MERS-CoV or genomic RNA significantly decreased, but these two vaccines did not provide sterilizing immunity, and signs of inflammation were observed in mouse lung tissue (34).
An inactivated rabies virus (RABV) expressing MERS S1 provided complete protection from MERS challenge in mice but three 10-μg doses of vaccine were needed (35). Furthermore, the Ad5/hDPP4-transduced mouse model used in these studies has limitations. Adenovirus (Ad5) expressing MERS S or S1 also induced a neutralizing antibody in mice (36).
Ad41, an enteric adenovirus, may induce enhanced mucosal immunity when administered via an oral or intragastric (i.g.) route (37, 38). However, i.g. immunization of both Ad41-S and Ad5-S failed to generate mucosal immunity. Although Ad41-S induced humoral immunity in serum, it was significantly less than Ad5-S (39).
Chimpanzee adenovirus-based vector systems have also been used (40). In our work, we demonstrated that a single dose as low as 104 PFU of PIV5-MERS-S was sufficient to provide 100% protection against lethal MERS-CoV challenge. The low dose is especially advantageous in a situation where a mass immunization program is needed in a short period of time. To the best of our knowledge, this is the most efficacious MERS-CoV vaccine tested in a relevant animal model.
The protective mechanism of PIV5-MERS-S vaccine in C57BL/6 mice is likely due to robust cellular immune responses after PIV5-MERS-S immunization. While neutralizing antibody was generated in C57BL/6 mice after a single-dose immunization with PIV5-MERS-S, titers were modest at 1:64 and 1:128 with 104 PFU and 106 PFU of PIV5-MERS-S, respectively (Fig. 2A and B).
Consistent with protective cellular immune responses protecting the mice, a significant influx of CD8+ IFN-γ+ cells was detected in lungs of C57BL/6 mice following PIV5-MERS-S immunization (Fig. 3). Furthermore, the observation that PIV5-MERS-S-immunized mice had a higher rate of MERS virus clearance (Fig. 4C) suggests a role for T cell-based immunity in protecting C57BL/6 mice against lethal challenge.
Interestingly, in BALB/c mice, PIV5-MERS-S generated neutralizing antibody titers as high as 1:2,000 (Fig. 2C). It is possible that the higher neutralizing antibody titers in BALB/c mice may be protective. Unfortunately, the only available small animal model is a humanized mouse model on the C57BL/6 background.
It is known that the S protein is a major protective antigen for coronaviruses. It may be possible to improve our vaccine efficacy by expressing additional MERS-CoV proteins such as N and M using PIV5 as a vector. However, a parainfluenza virus 3 (PIV3)-based SARS-CoV vaccine candidate expressing N, M, or E without the S protein failed to protect hamsters from SARS-CoV challenge (41).
The ability of PIV5-MERS-S to generate cellular and humoral immune responses in mice may be in part attributed to the ability of PIV5 to express the MERS S protein in its native conformation. As shown in Fig. 1C, PIV5-MERS-S caused massive syncytium formation in Vero cells, indicating the S protein was functional in promoting cell-to-cell fusion. Thus, we reasoned that the S protein produced in PIV5-MERS-S-infected cells maintains a native conformation.
The MERS S protein has 1,353 amino acid residues. The entire insertion of the S gene with proper regulatory sequences is over 4,000 nucleotides in length. This is the longest single gene we have inserted into the PIV5 genome. Since we inserted this gene between SH and HN, and the SH gene is not essential, it may be possible to remove SH to allow insertion of longer sequences. Thus, we speculate that the PIV5 genome can accommodate sequences longer than 4,000 nucleotides
It has been reported that inactivated SARS-CoV-immunized mice generated a hypersensitive-type lung pathology after virus challenge, raising the concern of vaccine-enhanced disease (42, 43). Previously, a formalin-inactivated, whole-virus respiratory syncytial virus (RSV) vaccine caused enhanced disease in vaccinated children, leading to vaccine-related deaths (44). Similarly, inactivated MERS-CoV has been reported to generate a Th2-type immunopathology after MERS-CoV challenge in mice (29).
In the case of a PIV5-based RSV vaccine, extensive studies indicate that PIV5-based RSV vaccine does not cause enhanced diseases (45). Thus, as a viral vector, PIV5 is not known to cause any enhanced diseases, and in our experiment, we observed no abnormal immune responses in PIV5-MERS-S-immunized mice after MERS-CoV challenge, suggesting that PIV5-MERS-S is unlikely to be associated with enhanced disease.
Lung tissues of mice immunized with inactivated MERS-CoV had an influx of eosinophils after MERS-CoV challenge, indicative of a hypersensitivity-type response. This result is consistent with a previous report that inactivated MERS-CoV immunization caused increased IL-4 and IL-5 expression and an influx of eosinophils in lungs after challenge (29).
Understanding whether immunization with inactivated MERS-CoV can cause enhanced disease is critical for developing a safe and effective vaccine.
While MERS-CoV has a high morbidity and mortality, it has very a low prevalence in human populations. Dromedary camels are considered the intermediate host that transmits MERS-CoV to humans. Thus, it may be possible to control the spread of MERS-CoV in humans by controlling infection in dromedary camels. Perhaps virus transmission from camels to humans can be blocked, with concomitant immunization of high-risk human populations, as proposed by CEPI (The Coalition for Epidemic Preparedness Innovations) and WHO.
As a vaccine vector, PIV5 has been effective in mice, cotton rats, hamsters, guinea pigs, ferrets, dogs, and nonhuman primates (25, 46–49). It will be worthwhile to test PIV5-MERS-S in camels in the future.
Recently, SARS-CoV-2 (2019-nCoV) was identified in Wuhan, China, in late 2019. This is a novel zoonotic CoV related to the SARS-CoV that can cause severe respiratory disease (COVID-19). To date, this virus resulted in a significant disease burden, with more than 465,000 cases reported in 199 countries and an estimated case fatality rate of ~2%.
The finding that PIV5 expressing MERS S protected mice against lethal MERS-CoV challenge at a single low dose of 104 PFU suggests its potential use as a vaccine vector for emerging viruses such as SARS-CoV-2. Further studies of using PIV5 expressing the S protein from SARS-CoV-2 as a vaccine candidate are ongoing.
reference link: https://journals.asm.org/doi/10.1128/mBio.00554-20
More information: Dong An et al, Protection of K18-hACE2 mice and ferrets against SARS-CoV-2 challenge by a single-dose mucosal immunization with a parainfluenza virus 5–based COVID-19 vaccine, Science Advances (2021). DOI: 10.1126/sciadv.abi5246
