key: cord-0945104-u99omznb authors: Jeyanathan, Mangalakumari; Afkhami, Sam; Smaill, Fiona; Miller, Matthew S.; Lichty, Brian D.; Xing, Zhou title: Immunological considerations for COVID-19 vaccine strategies date: 2020-09-04 journal: Nat Rev Immunol DOI: 10.1038/s41577-020-00434-6 sha: 6ba0a2f330a31d7879bbb0d5d246f660dec6fac4 doc_id: 945104 cord_uid: u99omznb The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the most formidable challenge to humanity in a century. It is widely believed that prepandemic normalcy will never return until a safe and effective vaccine strategy becomes available and a global vaccination programme is implemented successfully. Here, we discuss the immunological principles that need to be taken into consideration in the development of COVID-19 vaccine strategies. On the basis of these principles, we examine the current COVID-19 vaccine candidates, their strengths and potential shortfalls, and make inferences about their chances of success. Finally, we discuss the scientific and practical challenges that will be faced in the process of developing a successful vaccine and the ways in which COVID-19 vaccine strategies may evolve over the next few years. constitute a safe and immunologically effective COVID-19 vaccine strategy, how to define successful end points in vaccine efficacy testing and what to expect from the global vaccine effort over the next few years. This Review outlines the guiding immunological principles for the design of COVID-19 vaccine strategies and analyses the current COVID-19 vaccine landscape and the challenges ahead. Although much remains to be understood regarding the immune response to SARS-CoV-2, and vaccine-induced protective immunity may differ from natural immunity owing to the immune-evasion strategies of the virus, improved understanding of the natural immune response will be instrumental in developing effective vaccine and therapeutic strategies. It is particularly relevant to understand the difference in immune responses between asymptomatic, mild and severe cases and at early and late stages of infection, and to understand why seniors are particularly susceptible to COVID-19, whereas the young are better protected. It is estimated that 40-75% of infections may be mild or asymptomatic 7, 8 and asymptomatic individuals may have a significantly longer duration of viral shedding than their symptomatic counterparts 9 . Furthermore, that asymptomatic and mildly ill individuals seem to develop low levels of antibody-mediated immunity has important implications for understanding herd immunity. The initial site of infection of SARS-CoV-2 is the respiratory tract 10,11 . On entry, SARS-CoV-2 interacts with the angiotensin-converting enzyme 2 (ACE2) receptor Immunological considerations for COVID-19 vaccine strategies A rapidly developing lung condition characterized by deficient oxygen exchange and shortness of breath, resulting from severe lung injury and inflammation following infection. Age-related changes in the immune system that lead to a progressive reduction in its ability to develop effective antibody and cellular responses to infections and vaccinations. A persisting reset state of the innate immune system long after the initial antigen or microbial exposure that leads to enhanced responsiveness to the same or an unrelated antigen or microorganism. www.nature.com/nri that they may exert protection through Fc-mediated effector functions rather than direct virus neutralization 41, 42 . Somewhat unusually, several studies have reported that IgA responses to S protein peak earlier than IgM responses and are more pronounced, which makes IgA a potentially attractive target for antibody-based diagnostic assays 43, 44 . The mechanistic basis of this early induction of S-specific IgA is not yet clear. We do not yet understand the durability of the antibody responses to SARS-CoV-2. However, previous longitudinal studies of patients with SARS-CoV infection reported substantial waning of neutralizing antibody titres between 1 year and 2 years after infection 45, 46 . This is consistent with classical studies showing a relatively rapid waning of antibodies to the seasonal coronavirus 229E 47 . There are currently no immune correlates of protection for SARS-CoV-2 or other human coroanviruses. Thus, it is unclear what titre of neutralizing antibodies is sufficient to confer protection against infection. Establishing such correlates will be essential to guide the development of effective COVID-19 vaccines. T cell-mediated immunity. Whereas the current successful human antiviral vaccines, such as influenza and measles vaccines, depend largely on the induction of antibody responses, emerging evidence suggests the requirement of both antibody-mediated and T cellmediated immunity for effective protection against SARS-CoV-2 (reFs 17, 27 ). It is well known that CD4 + T cell help is important for optimal antibody responses and for CD8 + T cell activation in host defence 48 . Furthermore, if neutralizing antibody-mediated protection is incomplete, cytotoxic CD8 + T cells are crucial for viral clearance 49 . One study found that among people who had recovered from COVID-19, 100% had S protein-specific CD4 + T cells in the circulation and 70% had S proteinspecific CD8 + T cells in the circulation 30 , and preclinical studies show a protective role of T cells in host defence against SARS-CoV 50 . The 2-12-day incubation or presymptomatic period of SARS-CoV-2 infection is associated not only with virusmediated innate immune suppression but also with delayed activation of T cells, particularly CD8 + T cells 18, 19 , as is the case for SARS and MERS. People who have recovered from COVID-19 seem to have high levels of both neutralizing antibodies and T cells, and, compared with severe cases, milder cases of COVID-19 have greater numbers of memory CD8 + T cells in the respiratory tract 24, 29, 30 . Evidence suggests that the induction of such lung tissue-resident memory T cells (T RM cells) will depend on the route of vaccination. respiratory mucosal vaccination induces strong lung T RM cell responses, whereas parenteral vaccination fails to do so 51-53 . Experimentally, the airway T RM cells elicited by respiratory mucosal vaccination offered robust protection against SARS-CoV infection 54 . The T helper cell (T H cell) phenotype of vaccineinduced T cells is also relevant to the protection they mediate. Less severe cases of SARS were associated with accelerated induction of a T H 1 cell response 55 , whereas T H 2 cell responses have been associated with enhancement of lung disease following infection in hosts parenterally vaccinated with inactivated SARS-CoV viral vaccines 56,57 . Thus, COVID-19 vaccine-induced T RM cells should have a T H 1 cell-like phenotype. These lines of evidence, together with data suggesting that T cell-mediated immunity generally is a more reliable correlate of vaccine protection than antibody titres in seniors 26 , strongly support the inclusion of T cell responses in COVID-19 vaccine design 17,27 . Pre-existing cross-reactive immunity. Emerging evidence indicates that CD4 + T cells in 35% of healthy individuals not exposed to SARS-CoV-2 recognize the SARS-CoV-2 S protein and that CD4 + T cells in 40-60% of unexposed individuals are reactive to SARS-CoV-2 proteins other than S protein 30, 58 . This indicates that there is cross-reactivity between CD4 + T cells specific for SARS-CoV-2 and CD4 + T cells specific for human common cold coronaviruses, SARS-CoV and animal betacoronaviruses 17,59-61 . There are four human coronaviruses -229E, NL63, OC43 and HKU1 -that account for ~15% of common colds in humans 17 . Adults may be infected with one of these on average every 2-3 years, such that there could be a degree of pre-existing crossreactive immunity to SARS-CoV-2 antigens in these people, which offers a potential explanation for differing susceptibility to SARS-CoV-2 infection. In addition to understanding the relationship between such preexisting immunity to human coronaviruses and host defence against SARS-CoV-2, it will also be important to consider the contribution of COVID-19 vaccine-boosted cross-reactive immune responses to vaccine-induced protective immunity. innate immune memory (also known as trained immunity) is a recently recognized component of immunological memory that has implications for vaccine strategies 83, 84, 168, 169 . several live attenuated human vaccines induce trained immunity that can mediate non-specific protective responses to heterologous infections in addition to pathogenspecific adaptive immune memory [168] [169] [170] . the most well-studied human vaccine that induces trained immunity is the bacillus Calmette-Guérin (BCG) vaccine against tuberculosis 171 . BCG vaccination endows circulating monocytes with characteristics of trained immunity through epigenetic and metabolic rewiring of myeloid progenitors in the bone marrow 169, 172, 173 . these trained monocytes enhance protection against heterologous infections, including respiratory viral infection [174] [175] [176] . BCG may therefore offer a level of protection from coronavirus disease 2019 (COviD-19), which might be supported by the observed inverse correlation between universal BCG vaccination and COviD-19 fatalities 177 . several clinical trials are under way to assess the effects of BCG or measles vaccination on COviD-19 (reF. 178 ). a COviD-19 vaccine that can induce trained immunity might enhance early viral control by overcoming virus-imposed innate immune suppression and facilitating adaptive immune activation. the early timing of action by trained immunity is of importance as the overproduction of cytokines by macrophages at later stages of COviD-19 can contribute to immunopathology. although it remains to be understood how to best harness trained immunity for COviD-19 vaccine strategies, recent evidence suggests that routes of microbial exposure or vaccination determine the tissue distribution of trained immunity 83, 84, 169 . as respiratory mucosal immunity is key to early clearance of severe acute respiratory syndrome coronavirus 2 (sars-Cov-2), inducing trained immunity in alveolar macrophages and other innate cells 83, [179] [180] [181] through respiratory mucosal vaccination could be an effective strategy. indeed, a human serotype 5 adenovirus-vectored vaccine delivered to the respiratory mucosa induces memory alveolar macrophages capable of trained immunity against heterologous infections 85 . However, it is unclear whether lung memory macrophages may be replaced by inflammatory monocytes in response to sars-Cov-2. Fc-mediated effector functions immune functions that are mediated through the interaction of the constant Fc region of antibodies with innate immune molecules, complement proteins and specialized Fc receptors expressed by innate immune cells. The resulting functions include complementdependent cytotoxicity and antibody-dependent cellular phagocytosis or cell-mediated cytotoxicity. Direct administration of a vaccine to the respiratory tract by either intranasal delivery or aerosol inhalation. Administration of a vaccine via the skin, muscle or blood vessel. Importantly, whereas CD4 + T cells from patients with COVID-19 equally recognize the S1 and S2 subunits of SARS-CoV-2, cross-reactive CD4 + T cells from unexposed individuals recognize the S2 subunit 58 . CD4 + T cells from patients with COVID-19 cross-react strongly with S2 subunits of the human coronaviruses OC43 and 229E. More than 90% of tested healthy adults also have IgG antibodies specific for all four human common cold coronaviruses 17 . However, similarly to antibody responses to SARS-CoV and SARS-CoV-2, antibody responses to human coronaviruses wane rapidly within months after infection. Therefore, control of reinfection with human coronaviruses seems mainly to be antibody independent but T cell dependent 17 . As coronavirus cross-reactive T cells can be specific for both structural and non-structural viral proteins 58,61 , the extent of vaccine-boosted cross-reactive T cell responses induced by most protein subunit and recombinant viral-vectored COVID-19 vaccines, which are currently based only on the S protein, will be different from those boosted by multivalent COVID-19 vaccines such as those based on inactivated SARS-CoV-2 virus. One exception could be the use of live attenuated SARS-CoV-2 vaccines as the pre-existing cross-reactive immunity may limit the potency of such vaccines. Finally, it is noteworthy that the significant presence of cross-reactive immunity in some individuals calls for consideration of stratifying clinical trial participants receiving candidate COVID-19 vaccines according to their status of pre-existing coronavirus immunity. A potential barrier to the development of safe and efficacious COVID-19 vaccines (Box 2) is the risk that insufficient titres of neutralizing antibodies might trigger antibody-dependent enhancement (ADE) of disease. ADE is most classically associated with dengue virus, whereby cross-reactive but subneutralizing concentrations of antibodies to one virus serotype enhance infection with another serotype in Fcγ receptor (FcγR)-bearing cells, including macrophages 62 . A common property among viruses that cause ADE is an ability to replicate in macrophages and/or cause them to respond abnormally. Although macrophages do not seem to be a major target of SARS-CoV-2 infection, and the expression of ACE2 on different monocyte and macrophage populations is highly variable, previous data regarding SARS-CoV suggest that FcγRs can facilitate uptake of the virus into macrophages and B cells 21,63 . Cytokine profiles from patients infected with SARS-CoV-2 resemble those in macrophage activation syndrome and are characterized by high levels of inflammatory cytokines and chemokines 21,64-66 . Furthermore, patients with symptomatic COVID-19 are reported to produce IgG antibodies with reduced fucosylation levels, which in turn promotes their interaction with activating FcγRIIIa 67 . The evidence for ADE in the context of SARS-CoV infection is circumstantial. Correlations between antibody titres and infection severity have been reported, but it is unclear whether high antibody titres contribute to disease or whether severe infections elicit higher antibody titres 68 . Also, macrophages treated in vitro with serum from patients with SARS had exaggerated inflammatory cytokine profiles 69, 70 . ADE has been reported in some preclinical animal models vaccinated with experimental SARS-CoV vaccines. Ferrets vaccinated with a modified vaccinia virus Ankara (MVA) vaccine expressing full-length S protein had increased infection and hepatitis following challenge 71, 72 . Antibodies to S protein were reported to induce acute lung injury in experimentally infected macaques on the basis of histological examination 69 . By contrast, hamsters vaccinated with recombinant, full-length SARS-CoV S protein were protected against infection despite the ability of antibodies to mediate entry of SARS-CoV into B cells through FcγRII (reF. 73 ). Whether ADE occurs in the context of SARS-CoV-2 infection remains unclear but warrants further investigation, focusing directly on whether antibodies increase disease severity and, if so, characterizing the specific properties of these antibodies. What seems clear is that high levels of neutralizing antibodies can mediate protection. Defining the titres of neutralizing antibodies that are protective, ensuring that COVID-19 vaccines can achieve these titres and avoiding waning of antibodies to subneutralizing levels through frequent boosting will be important to minimize the possibility of ADE. Rationally designed COVID-19 vaccines that omit ADE-inducing, non-neutralizing or weakly neutralizing epitopes in favour of those known to mediate protective responses may also minimize the likelihood of disease enhancement. Finally, there is also evidence from mouse models of dengue virus infection that antiviral T cells help to dampen ADE of disease 74 . Therefore, a vaccine strategy designed to induce both neutralizing antibodies and robust T cell-mediated immunity may help to mitigate the risk of ADE. as most individuals infected with severe acute respiratory syndrome coronavirus 2 (sars-Cov-2) are asymptomatic or develop only mild symptoms and coronavirus disease 2019 (COviD-19) vaccines are being developed towards an ultimate goal of global mass immunization, vaccine safety is of paramount importance. any indication of a lack of safety consideration could also fuel the antivaccination movement and vaccine hesitancy, which would jeopardize the desired effect of achieving herd immunity. in this regard, most of the current COviD-19 vaccine clinical trials were initially conducted in healthy adults aged 55 years or younger, with only some later stage trials including seniors 98, 99, 115, [146] [147] [148] . the highly susceptible elderly populations and those with underlying medical conditions are in particular need of highly safe and effective vaccines. it remains largely unclear whether any of the initially trialled COviD-19 vaccines will be safe for both young children and seniors in both the short term and the long term. it remains likely that a different vaccine strategy with proven safety and efficacy profiles might be required for protection in seniors. the safety of a vaccine is generally determined by the nature of the vaccine platform, the choice of adjuvant, the mode and route of vaccine administration, the age of vaccinees and the status of pre-existing vaccine immunity 78 . For example, replicating live attenuated virus or viral-vectored vaccines may not be safe for a respiratory mucosal route of vaccination. Neither are certain immune adjuvants such as alum and bacterial-derived proteins. when a prime-boost immunization strategy is required, adverse events are generally more frequent and intense following the booster vaccination 115 . vaccine strategies for COviD-19, as for some other respiratory viral infections, require additional safety considerations related to the possibility of antibody-dependent enhancement of disease and the role of overproduction of proinflammatory cytokines in lung immunopathology. the latter pertains particularly to the application of respiratory mucosal vaccine strategies. (ADe). ADe of disease results when vaccine-induced non-neutralizing or weakly neutralizing antibodies bind to newly infecting virus to promote enhanced virus uptake into host cells via Fcγ receptors. This phenomenon has been observed experimentally or clinically following vaccination against viral pathogens such as dengue virus, respiratory syncytial virus and feline coronavirus. Also known as cytokine storm or secondary hemophagocytic lymphohistocytosis. A clinical state of systemic hyperinflammation that is characterized by hypercytokinaemia, fever, adenopathy, hepatosplenomegaly, cytopenias and activation of intravascular coagulation. Vaccine design Vaccine design concerns the selection of antigens, vaccine platforms, and vaccination routes and regimen. The choice of vaccine platform determines the relative immunogenic strength of vaccine-derived viral antigens, whether an immune adjuvant is required and the nature of protective immunity. These attributes also determine the suitability of a vaccine for a particular route of vaccination, and whether a prime-boost vaccination regimen is required to increase vaccine-mediated protective immunity and its durability. Furthermore, the selection of live attenuated viral vaccines or a respiratory mucosal route of vaccination will require more stringent safety testing (Box 2). The structural proteins present in the infectious virion include S protein, N protein, matrix (M) protein and envelope (E) protein. The N protein coats the large positive-stranded RNA genome, which is encased in a lipid envelope derived from the host cell membrane, into which the other three proteins (S, M and E) are inserted. In the case of SARS-CoV, it has been shown that only antibodies directed to S protein can neutralize the virus and prevent infection 75 . As a result, all SARS-CoV-2 vaccines in development include at least a portion of the S protein. These may be restricted to only the S1 domain or the RBD. Non-neutralizing antibodies to both S protein and the other exposed proteins (E and M) are generated. As there is a suspected role of these non-neutralizing antibodies, as well as weakly neutralizing antibodies, in ADE of disease, the inclusion of other structural (N) and/or non-structural proteins as vaccine antigens may help to create a more balanced response involving both humoral and T cell-mediated immunity. These could be highly expressed proteins such as N protein or highly conserved functional proteins that have a crucial role in the viral life cycle. For example, inclusion of viral enzymes such as the RNA-dependent RNA polymerase in a vaccine design may ensure that it targets all emerging variant strains, as these proteins are highly conserved 59,76,77 , even across other bat-derived coronaviruses that could emerge as a threat to humans in the future. Vaccine platforms. In general, vaccine platforms are divided into six categories: live attenuated virus, recombinant viral-vectored vaccines that are bioengineered to express target pathogen antigens in vivo, inactivated or killed virus, protein subunit vaccines, virus-like particles (VLPs) and nucleic acid-based (DNA or mRNA) vaccines. In broad terms, vaccines require two components: antigens from the target pathogen that are provided to or generated by the vaccine recipient; and an infection signal (such as a pathogen-associated molecular pattern or damage-associated molecular pattern) that alerts and activates the host immune system. Live attenuated vaccines can naturally provide both of these components, whereas non-viral vaccine platforms can provide the antigens but often require the artificial provision of signals to alert the immune system known as adjuvants. Typically, these non-viral vaccine platforms require multiple vaccinations to induce protective immunity, whereas live virus-based vaccines have the ability to provide 'one-shot' immunity. Similarly to non-viral platforms, killed virus vaccines sometimes require the inclusion of an adjuvant and repeated administration for full efficacy 78 . There are immunological pros and cons to each of these technologies as discussed later (TABle 1) . In addition to the careful selection of vaccine antigens and platform, the route of vaccination is an integral consideration of vaccine strategies 52,79 . This is particularly important for mucosal pathogens such as SARS-CoV-2 and those pathogens against which optimal protection requires not only neutralizing antibodies but also innate and adaptive cellular immunity 17, 80 . The best window of opportunity for SARS-CoV-2 control and clearance is the asymptomatic or presymptomatic period of COVID-19 (2-12 days), which is likely to require all of the immune protective elements to be present within the respiratory mucosa before viral entry 16,17,27 . The route of vaccination has a crucial role in determining this 52,81 . Protective IgG antibodies induced by parenteral vaccination readily appear at the respiratory mucosa, this being the primary mechanism by which intramuscular injection of measles or influenza vaccine offers protection in humans. However, this route of vaccination is unable to effectively induce mucosal IgA antibodies or T RM cells in the lungs 52,81 . By comparison, the respiratory mucosal route of vaccination is adept at inducing antibodies and T RM cells in the respiratory mucosa, as well as macrophage-mediated trained immunity 52,54,80-85 (Box 1). Inactivated virus, protein subunit and nucleic acid vaccines cannot be administered by the respiratory mucosal route owing to their requirement for potentially unsafe immune adjuvants and repeated delivery (TABle 1) . By contrast, recombinant viral-vectored vaccines, particularly those using human serotype 5 adenovirus (Ad5) or chimpanzee-derived adenovirus (ChAd), are safe and highly effective for respiratory mucosal vaccination 79 . Often, weakly immunogenic vaccines based on inactivated virus, protein subunits, nucleic acids or viral vectors such as Ad26 require a repeated homologous vaccination regimen to be effective. Indeed, most current human vaccines require repeated doses. As it is not yet known which COVID-19 vaccine strategy will be used or for how long the vaccine-induced protection may last in humans, it remains possible that a homologous or heterologous prime-boost vaccination regimen will be required to sustain protection, even with robust stand-alone platforms such as ChAd. The same or a different route may be used for the repeated vaccine delivery. Major COVID-19 vaccine candidates As of 31 July 2020, there were 27 vaccine candidates for COVID-19 in clinical evaluation and 139 vaccines in preclinical development 5 (Fig. 1 ). Of the 27 vaccines undergoing clinical evaluation (TABle 2) , the three lead candidates are viral-vectored and mRNA-based Virus-like particles (VlPs). A type of subunit vaccine based on multiple virus-derived proteins that are assembled to mimic the organization and conformation of authentic native viruses but that lack the viral genome. Biochemical components additional to vaccine antigens that are included in a vaccine formulation to help stimulate an adaptive immune response to vaccine antigens by activating innate immune cells. often, non-live vaccines such as inactivated virus, protein subunit and nucleic acid vaccines require immune adjuvants. A repeated immunization regimen designed to increase and sustain vaccine-induced immune responses. it may involve repeated delivery of the same vaccine (homologous) or sequential delivery of different vaccine platforms (heterologous). Live attenuated viral vaccines. Historically, several successful human vaccines, such as measles vaccine and the bacillus Calmette-Guérin (BCG) vaccine for tuberculosis (TB), have been based on attenuated strains of the actual pathogen 86 , with loss or mutation of virulence genes through in vitro passage. It is now possible to rationally design attenuated virus strains by mutating or deleting virulence genes. These deletion mutants can often replicate to a limited extent in host cells but lose the ability to cause disease in vivo. Coronaviruses have several genes that are not required for replication and that can be deleted, leading to attenuation in vivo. Deletion of various non-structural proteins, as well as of the structural E protein, has been used as a strategy to engineer vaccine strains of several zoonotic and veterinary coronaviruses [87] [88] [89] . Deletion of the E protein leads to attenuation and generation of an efficacious vaccine strain 87, 88 , but reversion of the attenuated phenotype has been reported 90 . Deletion of virulence factors may therefore provide a preferred mechanism of attenuation. For example, deletion of the 2′-O-methylase gene from the SARS-CoV genome removes the ability of the virus to hide its RNA from the host cell proteins MDA5 (also known as IFIH1) and IFIT1, thereby inducing a robust antiviral response in vivo 91 . Another approach to viral attenuation is known as codon deoptimization, whereby the nucleic acid sequence is modified to use suboptimal codons to encode the wild-type amino acid sequence, which considerably slows the translation of the viral protein during infection. This approach can yield a virus that is highly attenuated in vivo but still able to replicate in vitro if the correct viral protein is selected for deoptimization 92, 93 . However, the generation of an attenuated strain of a pathogen for use as a vaccine requires demonstration of its inability to revert genetically to become pathogenic (TABle 1; Box 2). This is particularly challenging in the case of coronaviruses as they are known to recombine in nature 94 , and an attenuated vaccine strain could, in theory, recombine with wild coronaviruses to recreate a pathogenic strain. So far, there are only three attenuated SARS-CoV-2 vaccines generated by codon deoptimization under preclinical development, by Mehmet Ali Aydinlar University in Turkey, Codagenix and Serum Institute of India, and Indian Immunologicals Ltd and Griffith University 5 . Recombinant viral-vectored vaccines are built on either a replicationdeficient viral backbone or an attenuated replicationcompetent viral backbone that is bioengineered to Although only a couple of viral-vectored vaccines have been approved for human use for the control of infections such as Ebola, this platform has been widely investigated and has a well-established track record for infectious diseases and cancer, given its genetic malleability, safety and ability to induce strong T cell responses without the need for an adjuvant 95, 96 . Some viral vectors, such as Ad5 and ChAd, usually need to be administered only once for protection and have natural tropism for the respiratory mucosa, which means they are amenable to respiratory mucosal vaccination 79 . The technology already exists for their large-scale clinical grade production and storage. Thus, recombinant viral vectors are the second most common platform for COVID-19 vaccine development, with 4 candidates currently in clinical trials (TABle 2) , 38 under preclinical development 5 and 3 (ChAdOx1 nCoV-19, Ad26-S and VSV-S) selected for Us operation Warp speed 97 (TABle 2) . The non-replicating viral platforms are mostly based on Ad5 or MVA, and most of these vaccine candidates express the S protein or RBD of SARS-CoV-2. Replication-competent viral vectors are mainly based on the vaccine strains of other human pathogens (such as measles or influenza viruses) or veterinary pathogens (such as vesicular stomatitis virus (VSV)). However, it will be important to consider whether humans have pre-existing immunity against the viral backbone (TABle 1) . Pre-existing antibodies can impair the ability of such vaccines to engage the immune system. Use of viral backbones such as ChAd, for which humans have little to no pre-existing immunity, can help to circumvent this issue 79 . Ad5-nCOV, which is being developed by the Chinese vaccine company CanSino Biologics, is designed to induce neutralizing antibodies to SARS-CoV-2 S protein following intramuscular injection (TABle 2) . Without published preclinical data, it entered phase I/II clinical trials with three doses of vaccine tested 98, 99 . Of note, these doses are 10-30 times higher than those used in previous trials of intramuscular vaccines [100] [101] [102] . Whereas the highest dose generated unacceptable toxicity and was dropped from the phase II study 99 , the smaller doses induced S protein-specific neutralizing antibodies in only 50% of the vaccine recipients 98 . The phase II study largely reaffirms the phase I observations that, although the vaccine induces both antibody and T cell responses, its potency is reduced by pre-existing immunity to Ad5, particularly in elderly participants 99 . Depending on geographical region, 35-95% of humans have significant circulating levels of neutralizing antibodies to Ad5 (reF. 103 ). This is consistent with the rapidly declining antibody titres observed in a phase II Ad5-Ebola vaccine study 104 . The vaccine is entering further advanced trials in China and Canada, but the efficacy of this strategy is now in question 105 . Another human adenovirus-based COVID-19 vaccine, known as Ad26-S, is being developed by Johnson & Johnson, although there is still 40% seroprevalence for Ad26 in humans 106 . As Ad26 is inherently less immunogenic than Ad5 (reF. 107 ), effective immunity requires repeated homologous or heterologous vaccination, as has been shown in Ad26-HIV and Ad26-Ebola vaccine studies in humans 108, 109 . Nevertheless, a single parenteral administration of an Ad26-vectored COVID-19 vaccine (Ad26.COV2.S) offered robust protection in a non-human primate model of SARS-CoV-2 (reF. 110 ). ChAdOx1 nCoV-19 (also known as AZD-1222), which is being developed by Oxford University, UK, and AstraZeneca, is the most clinically advanced COVID-19 vaccine (TABle 2) . Humans have low seroprevalence for ChAd, hence its strong immunogenicity and utility for heterologous prime-boost COVID-19 vaccination 79, 107, 111 . The development of ChAdOx1 nCoV-19 is based on promising human studies with ChAdOx1-MERS vaccine 112 and ChAdOx1-TB vaccine 113 . However, although intramuscular delivery of ChAdOx1 nCoV-19 reduced SARS-CoV-2 viral load in the lungs and prevented pneumonia in rhesus macaques, it did not reduce viral loads in the upper respiratory tract 114 . A recently reported phase I/II study shows its safety and the induction of potent neutralizing antibody and T cell responses following a single parenteral injection, which are boosted further by a second homologous vaccination 115 . It remains unclear from this trial to what extent both CD4 + and CD8 + T cell subsets were activated. VSV-S is a replication-competent COVID-19 vaccine under development by Merck 116 and other groups. Merck's vaccine is built upon the licensure of its highly efficacious VSV-Ebola vaccine, which induces neutralizing antibodies and cellular immunity against Ebola virus surface glycoprotein 117 Nature reviews | Immunology limited to 4 kb, and its suitability for respiratory mucosal vaccination is unclear. A single parenteral vaccination with a VSV vector expressing S protein provides protection against SARS-CoV-2 in both mouse and hamster models 118, 119 . Among other viral-vectored candidates is non-replicating MVA. MVA has widely been explored as a vaccine carrier and has a cloning capacity of up to 30 kb. However, as it is not robustly immunogenic, MVA is often used as a booster vaccine or repeated injection is required to be effective, as was the case in clinical testing of an MVA-MERS-S vaccine 120 . Physically or chemically inactivated viruses have been used successfully in human vaccines against polio, hepatitis A and influenza 121, 122 . Inactivated viruses can be rapidly generated and scaled up in a pandemic situation using well-established infrastructure and methods 123 . Inactivated viral vaccines have few safety concerns, unlike their live attenuated counterparts, and they express a wide range of native viral antigens, including surface antigens with retained epitope conformations that can induce conformation-dependent antibody responses 124, 125 . Currently, there are five early clinical trials to assess inactivated SARS-CoV-2 vaccines (TABle 2) , with an additional nine candidates in preclinical development 5 . PiCoVacc, an inactivated SARS-CoV-2 and alum-adjuvanted vaccine developed by Sinovac Biotech Ltd in China, is the most advanced candidate with published preclinical results 126 . It protects rhesus macaques against SARS-CoV-2, with reduced viral titres and immunopathology associated with antibodies to S protein and nucleocapsid 126 . BBIBP-CorV, another inactivated virus candidate, which is being developed by Chinese state-owned Sinopharm, was tested in a range of animal models, with demonstrated efficacy in non-human primates 127 . Although these findings provide optimism, the observations were made in rather short-term studies and should be interpreted with caution. Inactivated viral vaccines often require an adjuvant and repeated administration to be effective (TABle 1) . The use of alum as an adjuvant 126,127 makes them unsuitable for respiratory mucosal delivery 128 . Although the protection mediated by intramuscular immunization with PiCoVacc or BBIBP-CorV indicates some level of mucosal immunity, probably through the transport of systemic antibodies to the lungs, the durability of such immunity remains unclear as SARS-CoV-2 challenge was performed 1-4 weeks after vaccination 126, 127 . Furthermore, similarly to protein subunit vaccines, inactivated viral vaccines are poor inducers of cytotoxic CD8 + T cells, which are likely to be required for an effective COVID-19 vaccine. Studies with inactivated SARS-CoV and respiratory syncytial virus vaccines have reported vaccine-related enhancement of disease, likely involving a T H 2 cell response and lung eosinophilia, which may be worsened in aged hosts 56, 74, 129 . Although PiCoVacc or BBIBP-CorV did not worsen lung disease within 7 days after infection, alum is known to drive T H 2 cell-mediated immune responses, which warrants further safety investigations. The use of T H 1 cell-skewing modified alum or other adjuvants such as CpG may avert such safety concerns 130, 131 . Protein subunit vaccines. Currently, there are seven COVID-19 subunit vaccines in clinical trials (TABle 2) , with 50 other candidates under preclinical development, making this the most common platform 5 . Subunit vaccines primarily induce CD4 + T H cell and antibody responses. Therefore, most of these vaccines contain full-length SARS-CoV-2 S protein or portions of it with the goal of inducing neutralizing antibodies, similarly to the majority of SARS and MERS vaccines, which had differing levels of efficacy [132] [133] [134] . Subunit vaccines can be designed to focus the immune response towards neutralizing epitopes, thereby averting the production of non-neutralizing antibodies that may promote ADE of disease 135 . However, unlike nucleic acid-based or viral-vectored vaccines, recombinant S proteins in subunit vaccines could have an improper epitope conformation unless they are produced in mammalian cells 136 . Proteins or peptides alone are poorly immunogenic and generally require not only an adjuvant but also repeated administration, and they are poor activators of CD8 + T cell responses (TABle 1) . Furthermore, this platform is generally unsuitable for respiratory mucosal vaccination. As is the case for inactivated viral vaccines, use of unmodified alum as an adjuvant skews the immune response towards T H 2 cell-like responses 56 , which is undesirable for host defence against SARS-CoV-2 and may have a role in ADE of disease 74, 130 . In this regard, subunit COVID-19 vaccines being developed by GlaxoSmithKline and Novavax use AS03 and Matrix-M adjuvants, respectively 5 . there is an urgent need to identify suitable animal models for the preclinical evaluation of coronavirus disease 2019 (COviD-19) vaccines 182 . a large number of animal species have differing degrees of susceptibility to severe acute respiratory syndrome coronavirus 2 (sars-Cov-2) infection, depending on the relative binding affinity of the virus to the host angiotensin-converting enzyme 2 (aCe2) receptor or on host protease activities on the s protein 183 . among the animal species tested, aCe2 of rhesus macaques has the greatest binding activity for sars-Cov-2 (reF. 183 ). infected macaques shed sars-Cov-2 from the upper and the lower respiratory tract but they do not develop the same clinical signs and age-dependent disease severity as humans 184, 185 . Cats, ferrets and hamsters are also susceptible to sars-Cov-2 infection. Notably, natural airborne and contact transmissions of sars-Cov-2 have been reported in cats and hamsters, respectively, but not in ferrets 186 . Hamsters, but not cats and ferrets, manifest severe clinical symptoms. thus, these animal models are differentially capable of recapitulating relevant aspects of COviD-19. Mouse models are widely used for vaccine testing owing to their affordability and the availability of immunoreagents and transgenic mouse strains. However, the aCe2 of conventional mice does not bind well to sars-Cov s protein 187 . transgenic mice expressing human aCe2 were initially developed and thoroughly characterized for the study of sars-Cov and have now been shown to support sars-Cov-2 replication in the lung, and these mice develop interstitial pneumonia similar to humans 188 . Human aCe2-expressing mice that are further humanized to express human HLa genes and/or to have human immune cells will be useful for studying human immune responses and immunodominant epitopes following vaccination and viral infection with sars-Cov-2. Beyond animal models, of further relevance to human applications is the ongoing ethical debate regarding intentional challenge of vaccinated young people with sars-Cov-2. www.nature.com/nri Virus-like particles. VLPs are spontaneously forming particles composed of several structural viral proteins that are co-expressed or admixed. Several commercial vaccines, such as hepatitis B and human papillomavirus vaccines, are based on VLPs 137 . In the case of enveloped coronaviruses, VLPs form when the viral proteins S, M and E, with or without N, are co-expressed in eukaryotic producer cells 138, 139 . This results in active budding from the producer cells of VLPs that are structurally identical to the infectious virus but lack the viral genome and thus are non-infectious. The presence of S protein on the surface of VLPs enables them to bind and enter ACE2 + cells in the same manner as the parent virus 140 . Unlike subunit vaccines, the array of S protein on the VLP surface crosslinks the B cell receptor and directly activates B cells, but, like subunit and inactivated viral vaccines, VLPs also typically require an adjuvant and repeated administration 137 . Notwithstanding this, the VLP technology is well established, the biology and safety of coronavirus VLPs are understood and their large-scale production to Good Manufacturing Practice standards is relatively straightforward. Currently, there is only 1 VLP-based COVID-19 vaccine in clinical trials (TABle 2) , with 12 more under preclinical development 5 . These are produced either in vivo from a viral vector, such as MVA, that expresses the VLP components (a platform being developed by GeoVax) or more often in vitro from producer cells. Notably, Medicago, a Canadian company, produces its SARS-CoV-2 VLPs from genetically engineered plants. Its unpublished results seem to suggest efficacy in inducing neutralizing antibodies in mice 141 . Recombinant plasmid DNA has been explored as a vaccine platform for decades, whereas mRNA has emerged more recently as a promising platform 142, 143 . Currently, there are 6 mRNA-based COVID-19 vaccines and 4 DNA-based COVID-19 vaccines in clinical trials (TABle 2) , with 27 such vaccines (16 mRNA-based and 11 DNA-based vaccines) under preclinical development 5 . The antigen-encoding mRNA complexed with a carrier such as lipid nanoparticles can be efficiently delivered in vivo into the cytoplasm of host cells for protein translation and post-translational modifications 142, 144 , which is an advantage over recombinant protein subunit vaccines. mRNA vaccines are non-infectious and are synthesized by in vitro transcription, free of microbial molecules. These beneficial features differentiate mRNA vaccines from live attenuated viral vaccines, inactivated viral vaccines, subunit vaccines and recombinant viral-vectored vaccines in terms of safety, efficacy and issues of antivector immunity, enabling their rapid and inexpensive production and repeated vaccination 142 (TABle 1) . mRNA-1273, which is produced by Moderna, an American biotech company that has experience with mRNA-based MERS vaccines, encodes a prefusionstabilized SARS-CoV-2 S protein encapsulated in lipid nanoparticles. It entered clinical testing even before the release of preclinical data 145 . Recently published phase I clinical trial data indicate that low and medium doses of two repeated parenteral injections are generally safe and induce strong S protein-specific antibody responses and a primarily CD4 + T cell response in most trial participants 146 . Pfizer and BioNTech are also assessing an mRNA-lipid nanoparticle vaccine encoding the S protein RBD (known as BNT162b1) in humans, who developed robust S protein-specific antibody and CD4 + and CD8 + T cell responses following two repeated parenteral injections 147, 148 . The Pfizer/BioNTech and Moderna vaccines have both been selected for US Operation Warp Speed 97 (TABle 2) . Although no mRNA vaccine has yet been licensed for human use, their potential is supported by previous studies of influenza, rabies and Zika virus infections in animals [149] [150] [151] [152] [153] . For example, an mRNA vaccine for influenza virus induced long-term humoral immunity in young and aged mice 149 , and an mRNA vaccine for Zika virus induced both antibodies and cytotoxic CD8 + T cells in mice 154 . However, two clinical studies show disparities in the magnitude and longevity of immune responses induced by mRNA vaccines 152, 155 . Thus, although mRNA-based COVID-19 vaccines show promise from early clinical testing, questions remain about their protective efficacy in humans. It is also unclear whether mRNA vaccines are amenable to respiratory mucosal delivery. Plasmid DNA vaccines share several characteristics with mRNA vaccines, including safety, ease of production and scalability 156 . However, they are poorly immunogenic, requiring multiple doses and the addition of an adjuvant. Currently, there are four plasmid DNA-based COVID-19 vaccines in clinical testing (TABle 2) , with 11 more under preclinical development. INO-4800, a plasmid DNA vaccine expressing SARS-CoV-2 S protein, is being developed by the US biotech company Inovio Pharmaceuticals. A preclinical study in mice and guinea pigs examined the immunogenicity of this vaccine but did not provide any data pertaining to protection against challenge 157 . Two repeated injections of an S protein-expressing plasmid DNA vaccine resulted in robust protective immunity in rhesus macaques 158 . The world is in dire need of safe, effective COVID-19 vaccine strategies. Many laboratories and companies have scrambled to rapidly develop these vaccines, resulting in more than 160 vaccine candidates, with a handful having entered phase I, II and III clinical trials within a short period of 6 months. Although we are just beginning to understand COVID-19 and its vaccine requirements, most of the advanced vaccine platforms have been extensively explored for other infections and cancer 79, 95, 96, 159 . While it is important to pursue various vaccine strategies in parallel, it is equally important not to lose sight of this existing scientific knowledge to make well-informed decisions around which strategies to prioritize. The various vaccine platforms and strategies have their immunological pros and cons (TABle 1) , but modern immunological principles and data from prior studies of similar platforms lead us to surmise that a parenteral COVID-19 vaccine strategy capable of inducing a robust, durable response involving both neutralizing antibodies Nature reviews | Immunology and T cells should provide a significant level of protection. Almost all of the current vaccines in the human immunization programme are delivered via the skin or muscle, and most of the current COVID-19 vaccine strategies also focus on the parenteral route of vaccination (TABle 2) . We further surmise that a respiratory mucosal vaccine strategy capable of inducing these responses directly in the respiratory mucosa will be most effective in the early control or clearance of SARS-CoV-2. This is particularly relevant to high-risk elderly populations, who will require a particularly robust vaccine strategy. In this regard, a respiratory mucosal vaccine strategy for Other groups to consider for vaccination • Health-care workers • Individuals with co-morbidities • Seniors • Ethnic minorities A 'pandemic vaccine' or a vaccine fully validated from the 'rationalized vaccine pipeline' to be offered to high-risk populations first owing to limited supplies • The best vaccine strategies identified from the rationalized vaccine paradigm • Regional immunization owing to limited distribution • Worldwide immunization • SARS-CoV-2 exposed • Waning immunity • Individuals infected with SARS-CoV-2 but who developed poor immunity • Those who initially developed immunity but it waned very quickly In response to the urgent demand for a vaccine, more than two dozen candidate vaccines are advancing through clinical trials following an expedited pandemic vaccine development paradigm, with many steps of the development process occurring in parallel before a successful outcome of previous steps has been confirmed. Vaccine candidates will continue to be preclinically and clinically evaluated following conventional and/or rationalized vaccine development processes over the next few years. These efforts will evolve to meet the demands for vaccination in several likely scenarios that are predicted on the basis of sociopolitical challenges and the emerging data regarding the trajectory of the coronavirus disease 2019 (COVID-19) pandemic and the host response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One scenario is the priority vaccination of high-risk populations such as health-care workers, seniors, people with co-morbidities and ethnic minorities, who have been disproportionately affected by COVID-19, when vaccine supply is initially limited. Aside from these prioritized groups, it may also be necessary to consider that asymptomatic individuals, patients who have recovered from COVID-19 but generated poor immunity or whose immunity quickly waned, and individuals who received a rapidly developed 'pandemic' vaccine that provided suboptimal protection or rapidly waning immune responses may require a booster vaccination to ensure sufficient levels of population protection for herd immunity. Ultimately, regional, continental and global populations will be subject to mass vaccination programmes based on the extent of national and global vaccine distribution and also likely according to the relative regional severity of outbreaks. www.nature.com/nri COVID-19 may draw on the successful experience in respiratory mucosal delivery of influenza, measles and TB vaccines to humans [160] [161] [162] . Respiratory mucosal vaccination also has the advantages of being needle-free and requiring a much smaller dose than the parenteral route. However, compared with the parenteral route, fewer vaccine platforms are safe and effective for respiratory mucosal vaccination. Furthermore, the use of inhalational devices for respiratory mucosal delivery may potentially be a limiting factor for widespread application in resource-poor settings. According to the pandemic vaccine development paradigm (Fig. 2) , the conventional vaccine development milestones are compressed from a time frame of 10-15 years to 1-2 years, with overlapping preclinical, clinical and scale-up manufacturing processes occurring in parallel 6 . Owing to the accelerated development process, the interim data from ongoing clinical and preclinical vaccine studies are being published almost in real time. As a result, crucial information about the longevity and quality of vaccine-induced protective immunity is unavailable. As transmission rates and the numbers of new cases have reduced in many countries, it is uncertain whether the phase II and phase III studies of the front-runner candidates will reach a reliable conclusion with regard to their protective efficacy. Furthermore, these vaccine candidates have been studied in isolation, which makes it difficult to directly compare the effectiveness of different candidates. Thus, it would be premature to hail the safety and immunogenicity observed in COVID-19 vaccine trials as a real success. To a large extent, such outcomes could be anticipated from past studies testing the same platforms and delivery routes. Nevertheless, rapid deployment of a vaccine with preclinical efficacy data but limited clinical data to high-risk populations may be necessary (Fig. 2) . The evolving process of vaccine development will continue over the next few years until more clinical trials are completed, additional vaccine strategies are evaluated and host defence against SARS-CoV-2, including postinfection immunity, is better understood (Fig. 2) . Probably not until then will global mass immunization become a reality. It is possible that the populations that receive the first round of vaccines will have waning immunity and require boosting using improved second-generation COVID-19 vaccines. Furthermore, in addition to unexposed individuals, some individuals who have recovered from COVID-19 who develop poor or waning immunity may also require vaccination 163 . Given the challenges in resources, manufacturing and issues associated with distribution and regional protectionism, the implementation of vaccination programmes will likely be uneven, asynchronous and variableinvolving different vaccine platforms and strategies around the globe 164, 165 . In this regard, some resource-rich countries have already secured large numbers of doses of different candidate vaccines without knowing which one may prove effective. The heated debate has begun globally over who should be at the front of the line when vaccine supply is limited. The founding of the COVID-19 Vaccines Global Access (COVAX) Facility by Gavi, the Coalition for Epidemic Preparedness Innovations (CEPI) and the WHO is an attempt to garner resources and unite higher-and lower-income countries for the coordinated, rapid, transparent and equitable access to COVID-19 vaccines worldwide. World Health Organization. WHO coronavirus disease (COVID-19) dashboard Epidemiology of and risk factors for coronavirus infection in health care workers Estimating the effects of nonpharmaceutical interventions on COVID-19 in Europe High contagiousness and rapid spread of severe acute respiratory syndrome coronavirus 2. Emerg World Health Organization. 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The authors declare no competing interests. Nature Reviews Immunology thanks P. Klenerman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.