A recombinant herpes virus expressing influenza hemagglutinin confers protection and induces antibody-dependent cellular cytotoxicity

Despite widespread yearly vaccination, influenza leads to significant morbidity and mortality across the globe. To make a more broadly protective influenza vaccine, it may be necessary to elicit antibodies that can activate effector functions in immune cells, such as antibody-dependent cellular cytotoxicity (ADCC). There is growing evidence supporting the necessity for ADCC in protection against influenza and herpes simplex virus (HSV), among other infectious diseases. An HSV-2 strain lacking the essential glycoprotein D (gD), was used to create ΔgD-2, which is a highly protective vaccine against lethal HSV-1 and HSV-2 infection in mice. It also elicits high levels of IgG2c antibodies that bind FcγRIV, a receptor that activates ADCC. To make an ADCC-eliciting influenza vaccine, we cloned the hemagglutinin (HA) gene from an H1N1 influenza A strain into the ΔgD-2 HSV vector. Vaccination with ΔgD-2::HA_PR8 was protective against homologous influenza challenge and elicited an antibody response against HA that inhibits hemagglutination (HAI⁺), is predominantly IgG2c, strongly activates FcγRIV, and protects against influenza challenge following passive immunization of naïve mice. Prior exposure of mice to HSV-1, HSV-2, or a replication-defective HSV-2 vaccine (dl5-29) does not reduce protection against influenza by ΔgD-2::HA_PR8. This vaccine also continues to elicit protection against both HSV-1 and HSV-2, including high levels of IgG2c antibodies against HSV-2. Mice lacking the interferon-α/β receptor and mice lacking the interferon-γ receptor were also protected against influenza challenge by ΔgD-2::HA_PR8. Our results suggest that ΔgD-2 can be used as a vaccine vector against other pathogens, while also eliciting protective anti-HSV immunity.

Influenza remains a global health threat. Seasonal strains of influenza A and B cause an estimated 5 million cases of severe infections and 500,000 deaths per year (1). Influenza pandemics have caused even greater morbidity and mortality. During the H1N1 pandemic of 1918 to 1919, 500 million people, approximately one-third of the world’s population at that time, were estimated to have been infected with this strain, leading to 50 million deaths (2). The H1N1 pandemic of 2009 is estimated to have caused up to 575,000 deaths (2). Currently, three types of influenza vaccines are offered annually in the United States: a recombinant virus expressing influenza proteins, chemically inactivated virus, and live attenuated virus (3). Regardless of the vaccine type, multiple strains are included to increase the chances of developing sufficient protection against major circulating influenza strains. However, these vaccines primarily elicit a neutralizing antibody response that is sensitive to changes in the influenza virus due to antigenic drift and shift (4). Antigenic drift results from an accumulation of random mutations in influenza antigens, like hemagglutinin (HA), altering sites recognized by the immune system (4). Influenza A strains can also undergo antigenic shift, whereby two different influenza strains infect the same cell to form a reassortant virus with new antigenic properties (4). Due to limited immunity in the population, these new strains are highly virulent, causing widespread epidemics and disease (4). With antigenic drift and shift, vaccine-mediated protection against circulating strains has been insufficient (5). Influenza vaccines that elicit more robust and long-term protection are therefore needed. Notably, if an influenza vaccine with ≥75% efficacy were to be broadly used in the United States, an estimated 19,500 deaths a year could be prevented and direct healthcare costs reduced by $3.5 billion (6).For many years, efforts to improve influenza vaccines have focused on eliciting an immune response for full, broad protection against both circulating and future strains of the virus. These studies have shown that, in general, neutralizing antibodies are sufficient for homologous protection (7). However, achieving heterologous protection may require more broadly neutralizing antibodies or nonneutralizing antibodies able to activate effector immune cells (5). Previous studies have found that passively transferred nonneutralizing monoclonal antibodies can be potently protective in a mouse influenza challenge model (8–10). Several novel strategies have attempted to generate a nonneutralizing response against influenza. For example, vaccines have been created to specifically target the conserved stem region of HA (11–13).Nonneutralizing antibodies stimulate effector cell mechanisms, including antibody-mediated phagocytosis and antibody-dependent cellular cytotoxicity (ADCC), both of which require activation of the Fcγ receptors (FcγRs) (14). Specific isotypes of IgG antibodies are associated with FcγR modulation and subsequent ADCC activation, including the IgG1 and IgG3 subtypes in humans, as well as IgG2a and IgG2c subtypes in mice (15–19). IgG2a and IgG2c isotypes are functionally equivalent and mouse strain-dependent, with IgG2c present in C57BL/6J mice (20). Recent studies have demonstrated that natural infection by influenza and vaccination elicit nonneutralizing antibodies with effector functions that contribute to protection (5, 9, 21–27). In mouse and nonhuman primate challenge models, ADCC-mediating antibodies have demonstrated protection against both homologous and heterologous influenza challenge (9, 28).Recently, we developed a single-cycle herpes simplex virus (HSV) vaccine that completely protects against vaginal, skin, and ocular challenges by HSV-1 and HSV-2 (29, 30). Protection elicited by this vaccine, designated ΔgD-2 for its lack of the essential glycoprotein D (gD) gene, is transferable via passive infusion of immune sera to naïve wild-type mice but not to mice lacking the Fcγ common chain (30). The immune response elicited by ΔgD-2 primarily elicits nonneutralizing antibodies with high levels of FcγRIV-activating function.We asked whether ΔgD-2 could be used as a vaccine platform to induce broadly protective FcγRIV-activating antibodies against a heterologous antigen, such as influenza HA. In this study, we demonstrate that our recombinant vaccine, ΔgD-2::HA_PR8, elicits protection against influenza with a high proportion of FcγRIV-activating antibodies. Additionally, anticipating the use of ΔgD-2 as a vaccine vector against other pathogens, we tested whether our construct would still be protective in mice lacking interferon (IFN) function. Many humans have inborn errors in their IFN signaling pathways, leading to more lethal outcomes in infection (31). Patients with such deficiencies are disproportionately represented among HSV encephalitis cases and are often diagnosed only after presenting with serious symptoms (32–38). This at-risk population underscores the importance of eliciting protection against HSV in the absence of a functional IFN-α/β response. Additionally, many pathogens, such as dengue virus, require mouse models lacking IFN function, and for ease of testing, an efficacious vaccine should remain functional in these mice (39–41). In this study, we demonstrate that ΔgD-2 is a versatile, immunogenic vaccine vector that provides a strong FcγRIV-activating immune response against heterologous pathogens, while maintaining its protective benefit against HSV, in both wild-type and IFN-deficient mice.