Nikole L. Warner

In early 2024, clade 2.3.4.4b highly pathogenic avian influenza (HPAI) A H5N1 virus was detected in United States dairy cattle. Although so far the public health threat of contemporary clade 2.3.4.4b H5N1 virus strains remains low, continued circulation in mammals and frequent spillover into humans poses a threat of pandemic H5N1. The United States and other countries have stockpiled vaccines and have plans in place to rapidly produce additional vaccine doses should a pandemic H5N1 virus emerge. However, the continued antigenic drift of clade 2.3.4.4b H5N1 antigens compared with historical antigens used by stockpiled vaccines has raised questions of whether these vaccines will confer protection or whether stockpiles need to be updated. We recently evaluated a replicating RNA (repRNA) vaccine against lethal contemporary 2.3.4.4b H5N1 virus challenge in mice and found that a homologous, but not historical, H5 hemagglutinin (HA)–based vaccine conferred protection. Here, we further evaluated the protective capacity of a repRNA expressing the contemporary 2.3.4.4b HA or a repRNA expressing a historical H5 HA (A/Vietnam/1203/2004) in a recently developed lethal nonhuman primate (NHP) challenge model. We found that both vaccines conferred robust protection against lethal 2.3.4.4b H5N1 virus challenge, reducing viral loads and signs of respiratory illness. Our data show that the repRNA platform can elicit protective immunity against lethal influenza virus challenge in NHPs and that historical H5 HAs can elicit cross-protective immunity.

The Sudan virus (SUDV) outbreaks in Uganda in 2022 and 2025 created public health concerns in-country and the entire East African region. There are currently no licensed countermeasures against SUDV. We developed a SUDV vaccine candidate based on a nanocarrier (LION^TM) complexed with an alphavirus-based replicon RNA. Here, we compare the protective efficacy of the LION-SUDV vaccine either encoding the SUDV glycoprotein (GP) alone or in combination with the Ebola virus (EBOV) GP (LION-Combination). A LION-EBOV vaccine which is protective against EBOV was also included to determine the potential for cross-protection against SUDV infection. Single-dose vaccinations were conducted three weeks before challenge with a lethal dose of guinea pig-adapted SUDV using a female guinea pig disease model. We demonstrate 100% survival and protection with the LION-SUDV and the LION-Combination vaccines, while the LION-EBOV vaccine achieved 50% protection. Antigen-specific humoral responses correlate with decreased virus replication and survival. This result warrants further studies in larger animal species to ensure that protective efficacy is maintained with the single-dose LION-SUDV vaccine.

The ongoing circulation of influenza A H5N1 in the United States has raised concerns of a pandemic caused by highly pathogenic avian influenza. Although the United States has stockpiled and is prepared to produce millions of vaccine doses to address an H5N1 pandemic, currently circulating H5N1 viruses contain multiple mutations within the immunodominant head domain of hemagglutinin (HA) compared to the antigens used in stockpiled vaccines. It is unclear if these stockpiled vaccines will need to be updated to match the contemporary H5N1 strains. Here we show that a replicating RNA vaccine expressing the HA of an H5N1 isolated from a US dairy cow confers complete protection against homologous lethal challenge in mice. A repRNA encoding the HA of a clade 1 H5 from 2004 (A/Vietnam/1203/2004) as utilized by some stockpiled vaccines, confers only partial protection. Our data highlight the utility of nucleic acid vaccines to be rapidly updated to match emergent viruses of concern while demonstrating that contemporary bovine H5N1 viruses can evade immunity elicited by historical HA antigens.

In early 2024, clade 2.3.4.4b highly pathogenic avian influenza (HPAI) A H5N1 virus was detected in United States dairy cattle. While so far the public health threat of contemporary clade 2.3.4.4b H5N1 virus strains remains low, continued circulation in mammals and frequent spillover into humans poses a threat of pandemic H5N1. The United States and other countries have stockpiled vaccines and have plans in place to rapidly produce vaccines should a pandemic H5N1 virus emerge. However, the continued antigenic drift of clade 2.3.4.4b H5N1 antigens compared to historical antigens used by stockpiled vaccines has raised questions of whether these vaccines will confer protection or if stockpiles need to be updated. We recently evaluated a replicating RNA (repRNA) vaccine against lethal contemporary 2.3.4.4b H5N1 virus challenge in mice and found that a homologous but not historical H5 hemagglutinin (HA)-based vaccine conferred protection. Here we further evaluated the protective capacity of a repRNA expressing the contemporary 2.3.4.4b HA or a repRNA expressing a historical H5 HA (A/Vietnam/1203/2004) in a recently developed lethal non-human primate challenge model. We found that both vaccines conferred robust protection against lethal 2.3.4.4b H5N1 virus challenge, protecting against clinical disease and death, reducing viral loads and signs of respiratory illness. Our data show the repRNA platform can elicit protective immunity against lethal respiratory influenza disease and that historical H5 HAs elicit cross-protective immunity.

The recent emergence and rapid response to severe acute respiratory syndrome coronavirus 2 was enabled by prototype pathogen and vaccine platform approaches, driven by the preemptive application of RNA vaccine technology to the related Middle East respiratory syndrome coronavirus. Recently, the National Institutes of Allergy and Infectious Diseases identified nine virus families of concern, eight enveloped virus families and one nonenveloped virus family, for which vaccine generation is a priority. Although RNA vaccines have been described for a variety of enveloped viruses, a roadmap for their use against nonenveloped viruses is lacking. Enterovirus D68 was recently designated a prototype pathogen within the family Picornaviridae of nonenveloped viruses because of its rapid evolution and respiratory route of transmission, coupled with a lack of diverse anti-enterovirus vaccine approaches in development. Here, we describe a proof-of-concept approach using a clinical stage RNA vaccine platform that induced robust enterovirus D68–neutralizing antibody responses in mice and nonhuman primates and prevented upper and lower respiratory tract infections and neurological disease in mice. In addition, we used our platform to rapidly characterize the antigenic diversity within the six genotypes of enterovirus D68, providing the necessary data to inform multivalent vaccine compositions that can elicit optimal breadth of neutralizing responses. These results demonstrate that RNA vaccines can be used as tools in our pandemic-preparedness toolbox for nonenveloped viruses.

Epstein-Barr virus (EBV) is associated with several malignancies, neurodegenerative disorders and is the causative agent of infectious mononucleosis. A vaccine that prevents EBV-driven morbidity and mortality remains an unmet need. EBV is orally transmitted, infecting both B cells and epithelial cells. Several virally encoded proteins are involved in entry. The gH/gL glycoprotein complex is essential for infectivity irrespective of cell type, while gp42 is essential for infection of B cells. gp350 promotes viral attachment by binding to CD21 or CD35 and is the most abundant glycoprotein on the virion. gH/gL, gp42 and gp350, are known targets of neutralizing antibodies and therefore relevant immunogens for vaccine development. Here, we developed and optimized the delivery of several alphavirus-derived replicon RNA (repRNA) vaccine candidates encoding gH/gL, gH/gL/gp42 or gp350 delivered by a cationic nanocarrier termed LION™. The lead candidate, encoding full-length gH/gL, elicited high titers of neutralizing antibodies that persisted for at least 8 months and a vaccine-specific CD8⁺ T cell response. Transfer of vaccine-elicited IgG protected humanized mice from EBV-driven tumor formation and death following high-dose viral challenge. These data demonstrate that LION/repRNA-gH/gL is an ideal candidate vaccine for preventing EBV infection and/or related malignancies in humans.

Dengue virus (DENV) is a major global health problem, with over half of the world’s population at risk of infection. Despite over 60 years of efforts, no licensed vaccine suitable for population-based immunization against DENV is available. Here, we describe efforts to engineer epitope-based vaccines against DENV non-structural protein 1 (NS1). NS1 is present in DENV-infected cells as well as secreted into the blood of infected individuals. NS1 causes disruption of endothelial cell barriers, resulting in plasma leakage and hemorrhage. Immunizing against NS1 could elicit antibodies that block NS1 function and also target NS1-infected cells for antibody-dependent cell cytotoxicity. We identified highly conserved regions of NS1 from all four DENV serotypes. We generated synthetic peptides to these regions and chemically conjugated them to bacteriophage Qβ virus-like particles (VLPs). Mice were immunized two times with the candidate vaccines and sera were tested for the presence of antibodies that bound to the cognate peptide, recombinant NS1 from all four DENV serotypes, and DENV-2-infected cells. We found that two of the candidate vaccines elicited antibodies that bound to recombinant NS1, and one candidate vaccine elicited antibodies that bound to DENV-infected cells. These results show that an epitope-specific vaccine against conserved regions of NS1 could be a promising approach for DENV vaccines or therapeutics to bind circulating NS1 protein.

Identifying the specific epitopes targeted by antibodies elicited in response to infectious diseases is important for developing vaccines and diagnostics. However, techniques for broadly exploring the specificity of antibodies in a rapid manner are lacking, limiting our ability to quickly respond to emerging viruses. We previously reported a technology that couples deep sequencing technology with a bacteriophage MS2 virus-like particle (VLP) peptide display platform for identifying pathogen-specific antibody responses. Here, we describe refinements that expand the number of patient samples that can be processed at one time, increasing the utility of this technology for rapidly responding to emerging infectious diseases. We used dengue virus (DENV) as a model system since much is already known about the antibody response. Sera from primary DENV-infected patients (n = 28) were used to pan an MS2 bacteriophage VLP library displaying all possible 10-amino-acid peptides from the DENV polypeptide. Selected VLPs were identified by deep sequencing and further investigated by enzyme-linked immunosorbent assay. We identified previously described immunodominant regions of envelope and nonstructural protein-1, as well as a number of other epitopes. Our refinement of the deep sequence-coupled biopanning technology expands the utility of this approach for rapidly investigating the specificity of antibody responses to infectious diseases.

Mammalian Arenaviruses are a geographically and genetically diverse family of viruses, which is separated into two sub-groups; the Old World (OW) and New World (NW) groups. Of the OW viruses, Lassa virus (LASV), found endemically in Western Africa, is an important human pathogen, causing hundreds of thousands of infections, and several thousand deaths annually. Interestingly, some villages in endemic regions, up to 45% of the population show seropositivity for the virus. It is hypothesized that seropositivity is a result of natural infection through inhalation or ingestion of infectious particles. However, the exact mechanism is still unknown. LASV’s natural reservoir is Mastomys natalensis, a common rat found in sub-Saharan Africa. Epidemiological studies have identified the inhalation, and/or ingestion of infectious rodent excreta as the primary route of transmission from rodent reservoir to human hosts. Additionally, controlled experiments investigating intragastric (i.g.) versus intravenous (i.v.) routes of inoculation of non-human primates (NHPs) have continued transmission through these routes. viii These studies utilized Lymphocytic Choriomeningitis Virus (LCMV)-WE, a strain of LCMV that results in Lassa Fever (LF)-like disease in NHPs, and LCMVArmstrong (ARM), a strain of LCMV that mimics subclinical infection. When administered i.v., LCMV-WE-infected NHPs became systemically infected, showing clinical signs much like that of LF, and died. However, when orally infected with this virus through i.g. inoculation, some of these animals recovered, and later, were protected from lethal doses of i.v. WE challenge. Due to the nature of natural transmission from rodent to humans, epithelial cells are amongst the first cells to come in contact with the virus. However, the role(s) of the epithelial barrier during these infections have yet to be investigated. In order to investigate the role of these cells during arenaviral infection, here, a cell culture model was developed to investigate the interaction of OW mammalian arenaviruses at the site of intragastric inoculation. An important finding of this works is that the patterns of entry and release are viral dependent, and attachment to epithelial surfaces may play a role in these phenomena. Furthermore, regardless of their pathogenic potential in NHPs, both strains of LCMV, as well as LASV’s close relative, MOPV, showed similar patterns of entry and release when exposed to the apical and basolateral surfaces of polarized intestinal epithelia. Additionally, the replication patters of vaccine candidate ML- 29; a reassortant virus that contains the L segment of MOPV, and S segment of LASV, providing the exact same GP1