Noroviruses, members of the Norovirus genus in the family Caliciviridae, are a group of nonenveloped, single-stranded, positive-sense RNA viruses. They are the most common viral pathogens causing epidemic and endemic acute gastroenteritis (AGE) with typical symptoms of watery diarrhea, vomiting, and stomach cramping, affecting humans in all age groups in both developing and developed countries. Noroviruses are highly contagious with a reproductive number of Ro > 2 [1]. They spread quickly through polluted water, food, and/or surfaces, often leading to large AGE outbreaks in a variety of closed and semi-closed settings, including long-term care facilities, schools, hospitals, military installations, and cruise ships. In addition, noroviruses are commonly responsible for community-acquired, sporadic diarrhea cases. It was estimated that norovirus infections cause approximately 700 million episodes of diarrhea globally, accounting for 20% of all diarrhea cases annually, with substantial morbidity and mortality [2]. In the U.S.A. alone, noroviruses cause around 20 million AGE incidents every year, leading to about 70,000 hospitalizations, and up to 800 deaths ^[3][4][3,4]. On a worldwide basis, the biggest norovirus disease burden occurs in low- and middle-income countries, claiming over 200,000 lives per annum with USD 4.2 billion in direct health system costs and USD 60.3 billion in social economic loss ^[5][6][5,6]. Thus, noroviruses represent a major global public health threat.

Since 2016, the WHO has recognized the development of a norovirus vaccine as a high priority [7]. However, the development of a broadly effective norovirus vaccine has been difficult, being affected by a few major factors. First, human noroviruses are genetically and antigenically diverse, consisting of five genogroups (GI, GII, GIV, GVIII, and GIX) which comprise 35 genotypes [8]. Multiple variants of various genotypes are often detected simultaneously. Even within a single genotype, such as the predominant genogroup II, genotype 4 (GII.4), new variants emerge periodically as a result of rapid evolution ^[9][10][11][9,10,11]. Second, noroviruses do not effectively grow in culture cells, making the conventional live attenuated or inactivated vaccine approaches unfeasible. Consequently, the nonreplicating, recombinant protein-based vaccine strategy, mostly using virus-like particles (VLPs), becomes a common choice. Third, the unavailability of a standard cell culture-based neutralization assay, an efficient animal model, and trustworthy immune corelates of norovirus protective immunity to evaluate vaccine candidates add further obstacles in the vaccine development.

Several major barriers were noted during the development of a norovirus vaccine. Noroviruses are known for their wide genetic and antigenic diversity. Infection by a single norovirus strain does not appear to confer a broad, long-lasting immunity ^[12][35]. The cross protection observed during the phase 2b field study of Takada’s vaccine may be due to previous exposures of the subjects to those genotypes ^[13][26]. Among the four norovirus vaccine candidates under clinical trials, the three bivalent vaccines contain similar antigen components of a GI.1 and a GII.4 strain (Table 1). These selections of antigens are based on the rationales that the predominant GII.4 genotype is responsible for most of the AGE burden of noroviruses [10] and that an inclusion of a GI strain could broaden the vaccine effectiveness because a previous study of a cocktail VLP vaccine candidate showed rapid rises in antibodies that blocked against norovirus VLPs of diverse genotypes binding to HBGA ligands ^[14][36]. On the other hand, since other non-GII.4 genotypes, such as GII.3 and GII.17, often contributed substantially to norovirus disease burden globally in the past ^[15][37], the Longkoma vaccine containing VP1 antigens of all four genotypes should provide a wider breadth of effectiveness compared with the bivalent vaccine candidates and, thus, may represent a future direction of norovirus vaccine development. In fact, GII.2 noroviruses were found to be predominant between 2016 and 2019 in China ^[15][16][17][37,38,39], suggesting that an even more complex panel of antigens representing more norovirus genotypes should be considered for even broader vaccine efficacy.

Company	Vaccine Candidate	Adjuvant	Administration Route	Antigen Format	Antigen Genotype	Status of Trial

Takeda	TAK-214	Chitosan/MPLA, aluminum salt	Intranasal, intramuscular	Noroviral VLP	GI.1/GII.4	Phase 2b clinical trial
Vaxart	VXA-NVV-104	Adenovirus expressing double-stranded RNAs	Oral	Adenovirus expressing noroviral VP1	GI.1/GII.4	Phase 1 clinical trial
NVSI	Hansenulapolymorpha	Aluminum salt	Intramuscular	Noroviral VLP	GI.1/GII.4	Phase 1 clinical trial
IPS/Zhifei	Longkoma	Aluminum salt	Intramuscular	Noroviral VLP	GI.1/GII.3/ GII.4/GII.17	Phase 2a clinical trial

Regarding the longevity of norovirus immune protection, limited studies showed that a natural infection resulted in protective immunity against homologous strains for a relatively short time, ranging from months to a few years ^[18][19][40,41]. Hence, further study of the immunology after norovirus infection and development of an approach to prolong the durability of the protective immunity after vaccination is important. Furthermore, reliable immune markers of norovirus immune protection need to be clearly defined. Current data suggest that mucosal IgA is a key factor to protect hosts from norovirus infection ^{[20][21][22][23]}[30,42,43,44], implying that the adenovirus vectored Vaxart vaccine that is designed to induce the mucosal IgA response locally in the intestine ^[24][32] can be a good approach for high vaccine efficacy. Several human challenge investigations indicated that serum IgA, memory B cell responses, and serum HBGA-blocking titers are also immune correlates of protection ^{[25][20][21][26]}[24,30,42,45]. By contrast, pre-existed serum IgG antibody levels seem not to play a protection role ^[12][35]. An epidemiological study even implied that serum norovirus-specific IgG titers were inversely related with protection against norovirus AGE ^[27][46], although information of norovirus genetic background and HBGA-related host susceptibility should also be taken into account in this context in future studies. Thus, it is essential to define and establish immunological correlates to truly indicate the protection of a norovirus vaccine. Based on our current understanding, a vaccine candidate that can stimulate broader immune responses to activate several lines of defense, including serum IgA, memory B-cell responses, and serum HBGA-blocking titers, will likely be a potent one. Finally, the lack of a cell culture-based neutralization assay and an effective animal model as tools to evaluate a norovirus vaccine represents other two challenges. In this context, the recent successes in the establishment of the human enteroid system ^[28][47] and the zebrafish larvae model ^[29][30][48,49] for norovirus replication have laid a solid foundation for future development of a conventional cell culture system and an effective animal model for noroviruses as useful tools to evaluate norovirus vaccines.

Norovirus infections induce both humoral and cellular immune responses. While antibody-mediated immunity is relatively well studied, our understanding of cellular immune response to human norovirus infection or vaccination remains limited ^[31][32][50,51]. As a result, most current clinical evaluations of norovirus vaccine candidates focus on the antibody-mediated immunity. A recent study ^[33][52] showed that GII.2 infection elicited broad antibody and cellular immunity activation for T cells, monocytes, and dendritic cells, offering a new paradigm and a future direction for norovirus vaccine development for broad immune response and protection.

In addition, the adjuvant is an important component of a successful vaccine to enhance the magnitude, breadth, and durability of the vaccine antigens. The currently tested adjuvants for norovirus vaccine candidates under clinical trials include aluminum salt for intramuscular delivery and a combination of chitosan and MPLA for intranasal administration of the VLP-based vaccines, as well as double-stranded RNAs for the adenovirus expressing VP1s. For future development, other adjuvants, particularly those that are licensed for use in human vaccines, including the oil-in-water emulsion adjuvant MF59, the Adjuvant Systems AS0 adjuvants, such as AS01, AS03, and AS04, and cytosine phosphoguanosine (CpG) 1018 (reviewed in ^[34][53]) should be tested for their potential to further improve the immunogenicity and protective efficacy of norovirus vaccines.

Finally, the current success of the mRNA vaccine platform in development of COVID-19 (coronavirus disease 2019) vaccines with high protective efficacy, as well as its further applications in developing vaccine candidates against influenza ^[35][54] and malaria ^[36][55], provide a new approach to advance the norovirus vaccine development program. Following the footsteps of COVID-19 vaccine development, noroviral VP1 or even its receptor binding protruding (P) domain that appears equivalent to the spike protein of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) should be a good target of an mRNA vaccine against noroviral infection.