ABSTRACT New approaches to vaccination are needed for protection against rapidly evolving viruses because emerging variant strains often evade neutralizing antibodies. Alternative strategies include targeting highly conserved antigens that change more slowly over time, which could provide the basis for universal vaccines. Recombinant adenovirus (rAd)-based vaccines expressing conserved influenza virus antigens nucleoprotein (NP) and matrix 2 (M2) provide powerful, long-lasting protection against challenge with diverse influenza strains, especially if administered intranasally. After intranasal administration of these rAd-based vaccines, animals are protected against morbidity and mortality in part by strong local T cell responses. However, concerns have been raised that these local responses could result in immune-mediated lung damage. In this study, we vaccinated mice intranasally with rAd vectors expressing influenza A/NP and/or M2, or influenza B/NP as a control, and then challenged them with an influenza A virus 15 months later. Protection persisted, as shown by accelerated viral clearance. Vaccination reduced the incidence and/or severity of histopathologic findings associated with influenza infection. Vaccination with A/NP+M2-rAd or A/NP-rAd reduced cytokine responses in the lungs following challenge. Granzyme B levels peaked and resolved earlier in animals vaccinated with A/NP+M2-rAd or A/NP-rAd than in other groups, consistent with a rapid CD8 T cell response that resolved more quickly. An influx of neutrophils, shown by a rise in neutrophil elastase, occurred in all groups following the challenge but returned to baseline more quickly in the two A/NP-vaccinated groups. Thus, vaccines inducing potent local immunity in the respiratory tract provided protection, rather than damage, to the lungs. IMPORTANCE Vaccines targeting highly conserved proteins can protect broadly against diverse viral strains. When a vaccine is administered to the respiratory tract, protection against disease is especially powerful. However, it is important to establish that this approach is safe. When vaccinated animals later encounter viruses, does reactivation of powerful local immunity, including T cell responses, damage the lungs? This study investigates the safety of mucosal vaccination of the respiratory tract. Non-replicating adenoviral vaccine vectors expressing conserved influenza virus proteins were given intranasally. This vaccine-induced protection persists for at least 15 months. Vaccination did not exacerbate inflammatory responses or tissue damage upon influenza virus infection. Instead, vaccination with nucleoprotein reduced cytokine responses and histopathology, while neutrophil and T cell responses resolved earlier. The results are promising for safe vaccination at the site of infection and thus have implications for the control of influenza and other respiratory viruses.

Universal influenza virus vaccines targeting antigens conserved among influenza A virus strains can protect from severe disease but do not necessarily prevent infection. Despite allowing low-level infection, intranasal immunization with adenovirus vectors expressing the conserved antigens influenza nucleoprotein (A/NP) and M2 reduces influenza virus transmission from vaccinated to unvaccinated contact mice.

Current influenza vaccines, live attenuated or inactivated, do not protect against antigenically novel influenza A viruses (IAVs) of pandemic potential, which has driven interest in the development of universal influenza vaccines. Universal influenza vaccine candidates targeting highly conserved antigens of IAV nucleoprotein (NP) are promising as vaccines that induce T cell immunity, but concerns have been raised about the safety of inducing robust CD8 T cell responses in the lungs. Using a mouse model, we systematically evaluated effects of recombinant adenovirus vectors (rAd) expressing IAV NP (A/NP-rAd) or influenza B virus (IBV) NP (B/NP-rAd) on pulmonary inflammation and function after vaccination and following live IAV challenge. After A/NP-rAd or B/NP-rAd vaccination, female mice exhibited robust systemic and pulmonary vaccine-specific B cell and T cell responses and experienced no morbidity (e.g., body mass loss). Both in vivo pulmonary function testing and lung histopathology scoring revealed minimal adverse effects of intranasal rAd vaccination compared with unvaccinated mice. After IAV challenge, A/NP-rAd-vaccinated mice experienced significantly less morbidity, had lower pulmonary virus titers, and developed less pulmonary inflammation than unvaccinated or B/NP-rAd-vaccinated mice. Based on analysis of pulmonary physiology using detailed testing not previously applied to the question of T cell damage, mice protected by vaccination also had better lung function than controls. Results provide evidence that, in this model, adenoviral universal influenza vaccine does not damage pulmonary tissue. In addition, adaptive immunity, in particular, T cell immunity in the lungs, does not cause damage when restimulated but instead mitigates pulmonary damage following IAV infection.IMPORTANCE Respiratory viruses can emerge and spread rapidly before vaccines are available. It would be a tremendous advance to use vaccines that protect against whole categories of viruses, such as universal influenza vaccines, without the need to predict which virus will emerge. The nucleoprotein (NP) of influenza virus provides a target conserved among strains and is a dominant T cell target. In animals, vaccination to NP generates powerful T cell immunity and long-lasting protection against diverse influenza strains. Concerns have been raised, but not evaluated experimentally, that potent local T cell responses might damage the lungs. We analyzed lung function in detail in the setting of such a vaccination. Despite CD8 T cell responses in the lungs, lungs were not damaged and functioned normally after vaccination alone and were protected upon subsequent infection. This precedent provides important support for vaccines based on T cell-mediated protection, currently being considered for both influenza and SARS-CoV-2 vaccines.

Current influenza vaccines rely on inducing antibody responses to the rapidly evolving hemagglutinin (HA) and neuraminidase (NA) proteins, and thus need to be strain-matched. However, predictions of strains that will circulate are imperfect, and manufacturing of new vaccines based on them takes months. As an alternative, universal influenza vaccines target highly conserved antigens. In proof of concept studies of universal vaccine candidates in animal models challenge is generally conducted only a short time after vaccination, but protective immunity lasting far longer is important for the intended public health impact. We address the challenge of providing long-term protection. We demonstrate here broad, powerful, and long-lasting immune protection for a promising universal vaccine candidate. A single intranasal dose of recombinant adenoviruses (rAd) expressing influenza A nucleoprotein (A/NP) and matrix 2 (M2) was used. Extending our previous studies of this type of vaccine, we show that antibody and T-cell responses persist for over a year without boosting, and that protection against challenge persists a year after vaccination and remains broad, covering both group 1 and 2 influenza A viruses. In addition, we extend the work to influenza B. Immunization with influenza B nucleoprotein (B/NP)-rAd also gives immune responses that last a year without boosting and protect against challenge with influenza B viruses of mismatched HA lineages. Despite host immunity to adenoviral antigens, effective readministration is possible a year after primary vaccination, as shown by successful immunization to a transgene product the animals had not seen before. Protection against challenge with divergent and highly pathogenic A/H7N9 virus was weaker but was enhanced by a second dose of vaccine. Thus, this mucosal vaccination to conserved influenza antigens confers very long-lasting immune protection in animals against a broad range of influenza A and B viruses.

The prospect of universal influenza vaccines is generating much interest and research at the intersection of immunology, epidemiology, and viral evolution. While the current focus is on developing a vaccine that elicits a broadly cross-reactive immune response in clinical trials, there are important downstream questions about global deployment of a universal influenza vaccine that should be explored to minimize unintended consequences and maximize benefits. Here, we review and synthesize the questions most relevant to predicting the population benefits of universal influenza vaccines and discuss how existing information could be mined to begin to address these questions. We review three research topics where computational modeling could bring valuable evidence: immune imprinting, viral evolution, and transmission. We address the positive and negative consequences of imprinting, in which early childhood exposure to influenza shapes and limits immune responses to future infections via memory of conserved influenza antigens. However, the mechanisms at play, their effectiveness, breadth of protection, and the ability to “reprogram” already imprinted individuals, remains heavily debated. We describe instances of rapid influenza evolution that illustrate the plasticity of the influenza virus in the face of drug pressure and discuss how novel vaccines could introduce new selective pressures on the evolution of the virus. We examine the possible unintended consequences of broadly protective (but infection-permissive) vaccines on the dynamics of epidemic and pandemic influenza, compared to conventional vaccines that have been shown to provide herd immunity benefits. In conclusion, computational modeling offers a valuable tool to anticipate the benefits of ambitious universal influenza vaccine programs, while balancing the risks from endemic influenza strains and unpredictable pandemic viruses. Moving forward, it will be important to mine the vast amount of data generated in clinical studies of universal influenza vaccines to ensure that the benefits and consequences of these vaccine programs have been carefully modeled and explored.

Gene therapy is a rapidly evolving field in which a wide variety of therapeutic approaches are being considered and tested. In regulating product development in such an area, it is important to be flexible and attentive to practical limitations. Gene therapy products also present novel issues. One example is the potential for recombination. Recombination can occur between sequences in a vector and other elements in a packaging cell or producer cell. Investigators with products under development should be aware of this problem and consider alternative selection markers, especially since simply omitting ampicillin from production runs may lead to poor yields. Animal studies are usually needed and informative in testing of new products prior to human use, even though they provide imperfect predictions about effects in humans. Gene therapy clinical trials involve special unresolved issues, as well as usual issues of design and ethics affecting all trials.

Current approaches to influenza control rely on vaccines matched to viruses in circulation. Universal influenza vaccines would offer the advantage of providing broad protection against diverse strains of influenza virus. Candidate universal vaccines are developed using model systems, often testing in naïve animals. Yet the human population is not naïve, having varied immune histories that include exposure to viruses. We studied a candidate universal influenza vaccine (replication deficient adenoviruses expressing the conserved influenza A antigens NP and M2 [A/NP+M2-rAd]) given intranasally, the route previously shown to be most effective. To model recipients exposed to viruses, we used mice given rhinovirus (RV1B), respiratory syncytial virus (RSV-A2), influenza B virus, or influenza A virus before or after universal influenza vaccine. Vaccine performance was assessed by measuring immune responses to NP and M2, and monitoring weight loss and survival following influenza A challenge. Prior influenza A virus infection enhanced the response to the vaccine by priming to conserved influenza A antigens. RSV-A2 or RV1B had no effect on antibody responses to NP and M2 in serum. None of the viruses inhibited the ability of the vaccine to protect against influenza A virus challenge. The study demonstrates that the usefulness of this universal vaccine is not confined to the immunologically naïve and supports possible use in a human population with a varied history of respiratory infections.

Despite all we have learned since 1918 about influenza virus and immunity, available influenza vaccines remain inadequate to control outbreaks of unexpected strains. Universal vaccines not requiring strain matching would be a major improvement. Their composition would be independent of predicting circulating viruses and thus potentially effective against unexpected drift or pandemic strains. This commentary explores progress with candidate universal vaccines based on various target antigens. Candidates include vaccines based on conserved viral proteins such as nucleoprotein and matrix, on the conserved hemagglutinin (HA) stem, and various combinations. Discussion covers the differing evidence for each candidate vaccine demonstrating protection in animals against influenza viruses of widely divergent HA subtypes and groups; durability of protection; routes of administration, including mucosal, providing local immunity; and reduction of transmission. Human trials of some candidate universal vaccines have been completed or are underway. Interestingly, the HA stem, like nucleoprotein and matrix, induces immunity that permits some virus replication and emergence of escape mutants fit enough to cause disease. Vaccination with multiple target antigens will thus have advantages over use of single antigens. Ultimately, a universal vaccine providing long-term protection against all influenza virus strains might contribute to pandemic control and routine vaccination.

Transmission of influenza virus between susceptible hosts mediates spread of infection in the population and can occur via direct-contact or airborne routes. Mathematical models suggest that vaccines that reduce viral transmission from infected individuals could substantially reduce viral spread in an epidemic or pandemic, even if they do not completely protect against infection. Vaccines targeting conserved nucleoprotein (A/NP) and matrix 2 (M2) antigens of influenza virus do not completely prevent infection upon influenza virus challenge, but reduce viral replication, morbidity, and mortality. Using a mouse model of influenza virus transmission, we have previously shown that immunization with recombinant adenovirus vectors expressing the combination of A/NP and M2 can reduce viral transmission to unimmunized contacts. Here we demonstrate that transmission reduction is more effective when mice are immunized against A/NP and M2 intranasally than via the intramuscular route. We show that immunization against the combination of A/NP and M2 is more effective at reducing transmission than either antigen alone, with a clear hierarchy of effectiveness (A/NP + M2 > A/NP > M2). Transmission reduction is seen to a similar degree under both direct-contact and airborne transmission conditions. Finally, using seroconversion as an indicator of infection, we show that immunizing contact mice against A/NP and M2 prevents a significant fraction (similar to 50%) from becoming infected under direct-contact conditions. These findings suggest that when strain-matched vaccines are unavailable, conserved antigen vaccines could not only reduce severity of disease in vaccinated individuals but also limit the spread of virus during influenza epidemics or pandemics. (C) 2018 Elsevier Ltd. All rights reserved.

Universal influenza vaccines are designed to protect against diverse strains of influenza virus. Preclinical testing of new vaccine candidates is usually done in naïve animals, despite intended use in the human population with its varied immune history including responses to previous vaccinations. As an approach more relevant to human use, we tested a candidate universal influenza vaccine in mice with a history of conventional vaccination. Female BALB/c mice were given two intramuscular doses of inactivated influenza vaccine (IIV) or diphtheria and tetanus toxoids vaccine (DT), one month apart. Another group was given two intranasal doses of live attenuated influenza virus (LAIV). One month after the second dose, mice were given the universal influenza vaccine: recombinant adenoviruses expressing influenza A nucleoprotein (A/NP) and matrix 2 (M2) (A/NP + M2-rAd). Immune responses to universal vaccine antigens A/NP and M2 were assessed by ELISA and interferon-γ ELISPOT. Protection was tested by challenge with mouse-adapted A/FM/1/47 (H1N1) and monitoring for weight loss and survival. Universal vaccine performance was enhanced, inhibited or unaffected by particular prior vaccinations. Mice given Afluria IIV and LAIV had greater antibody and T-cell response to A/NP than mice without prior vaccination, providing examples of enhanced A/NP + M2-rAd performance. Though Fluvirin IIV partially inhibited, the universal vaccine still provided considerable protection unlike conventional vaccination. Fluzone IIV and DT had no effect on A/NP + M2-rAd performance. Thus our results demonstrate that universal vaccine candidate A/NP + M2-rAd was at least partially effective in mice with diverse prior histories. However, the degree of protection and nature of the immune responses may be affected by a history of conventional vaccination and suggests that performance in humans would be influenced by immune history.

Abstract Universal influenza vaccines are designed to protect against diverse strains of influenza virus. In a pandemic, universal vaccines could be life-saving. Candidate vaccines are usually tested in naïve animals, despite intended use in the human population that has a varied immune history, including responses to vaccination. To be more relevant to humans, we tested a candidate universal influenza vaccine in mice with a history of conventional vaccination. Female BALB/c mice were given two intramuscular doses of inactivated influenza vaccine (IIV) or diphtheria and tetanus toxoids vaccine (DT), one month apart. Another group had two intranasal (i.n.) doses of live attenuated influenza virus (LAIV). One month after the second dose, mice were given the universal vaccine, recombinant adenoviruses expressing influenza A NP and M2 (rAd-NP+M2, i.n.). Antibody responses to universal vaccine antigens NP and M2 were assessed by ELISA. T-cell responses to antigens were determined by IFN-γ ELISPOT. Mice were challenged with mouse-adapted A/FM/1/47 (H1N1) and subsequent weight loss determined rAd-NP+M2 efficacy. Efficacy was enhanced, inhibited or unaffected by prior history. Mice given Afluria IIV and LAIV lost less weight and had a greater antibody and T-cell response to NP than mice without prior vaccination, which are examples of enhanced rAd-NP+M2 efficacy. Inhibited vaccine efficacy was observed in mice that received Fluvirin IIV and mice had low IgG2a antibodies to NP and M2. Fluzone IIV and DT had no effect on rAd-NP+M2 efficacy. This study demonstrates that the protection by the universal influenza vaccine may be affected by a history of conventional vaccination, and suggests that performance in humans would be influenced by immune history.

Abstract Background Antibody and T-cell immunity to conserved influenza virus antigens can protect animals against infection with diverse influenza strains. Although immunity against conserved antigens occurs in humans, whether such responses provide cross-protection in humans and could be harnessed as the basis for universal influenza vaccines is controversial. The 2009 pandemic provided an opportunity to investigate whether pre-existing cross-reactive immunity affected susceptibility to infection. Methods In 2009, we banked sera and peripheral blood mononuclear cells (PBMC) from blood donors, then monitored them for pandemic influenza infection (pH1N1) by polymerase chain reaction or seroconversion. Antibodies to hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix 2 (M2), and HA-pseudotypes were measured in sera. T-cell inteferon-γ enzyme-linked immunospot responses were measured in PBMC. Results There were 13 infections in 117 evaluable donors. Pre-existing T-cell reactivity to pH1N1 was substantial (of 153 donors tested, 146 had >100 spot-forming cells/106 cells). Antibodies reactive with pH1N1 were common: anti-NP (all donors) and anti-M2 (44% of donors). Pseudotype-neutralizing antibodies to H1 were detected, but not to highly conserved HA epitopes. Unexpectedly, donors with symptomatic pH1N1 infection had sharp rises in HA pseudotype-neutralizing antibodies, not only pH1N1 but also against multiple seasonal H1s. In addition, an exploratory study of a T-cell marker (response to NP418-426) identified probable infection missed by standard criteria. Conclusions Although the number of infections was inadequate for conclusions about mechanisms of protection, this study documents the wide variety of pre-existing, cross-reactive, humoral and cellular immune responses to pandemic influenza virus antigens in humans. These responses can be compared with results of other studies and explored in universal influenza vaccine studies.

A change in the properties of influenza virus in 1968 has left a profound mark on population immunity

Influenza has a major impact on the elderly due to increased susceptibility to infection with age and poor response to current vaccines. We have studied universal influenza vaccine candidates based on influenza A nucleoprotein and matrix 2 (A/NP+M2). Long-lasting protection against influenza virus strains of divergent subtypes is induced, especially with mucosal immunization. Here, we tested universal vaccination in BALB/c mice of different ages. Vaccination used intramuscular DNA priming to A/NP+M2 followed by intranasal (i.n.) boosting with recombinant adenoviruses (rAd) expressing the same antigens, or only A/NP + M2-rAd given i.n. Antigen-specific systemic antibody responses were induced in young, middle-aged, and elderly mice (2, 11-17, and 20 months old, respectively), but decreased with age. Antibody responses in bronchoalveolar lavage (BAL) were detected only in young mice. Antigen-specific T cell responses were seen in young and middle-aged but not elderly mice. A/NP+M2 vaccination by the two regimens above protected against stringent challenge in young and middle-aged mice, but not in elderly mice. However, mice vaccinated with A/NP-rAd or A/M2-rAd during their youth were partially protected against challenge 16 months later when they were elderly. In addition, a regimen of two doses of A/NP+ M2-rAd given i.n. one month apart beginning in old age protected elderly mice against stringent challenge. This study highlights the potential benefit of cross-protective vaccines through middle age, and suggests that their performance might be enhanced in elderly individuals who had been exposed to influenza antigens early in life, as most humans have been, or by a two-dose rAd regimen given later in life.

Linker for activation of T cells (LAT) is critical for the propagation of T-cell signals upon T-cell receptor (TCR) activation. Previous studies demonstrated that substitution of LAT lysines with arginines (2KR LAT) resulted in decreased LAT ubiquitination and elevated T-cell signaling, indicating that LAT ubiquitination is a molecular checkpoint for attenuation of T-cell signaling. To investigate the role of LAT ubiquitination in vivo, we have generated transgenic mice expressing WT and ubiquitin-defective 2KR LAT. On TCR stimulation of T cells from these mice, proximal signaling and cytokine production was elevated in 2KR versus wildtype (WT) LAT mice. Enhanced cytolytic activity as well as T-helper responses were observed on LAT expression, which were further elevated by 2KR LAT expression. Despite greater T-effector function, WT or 2KR LAT expression did not have any effect on clearance of certain pathogens or tumors. Our data support the model that lack of tumor clearance is due to increased differentiation and acquisition of effector phenotype that is associated with suboptimal immunity in an immunotherapy model. Thus, our data further reinforce the role of LAT ubiquitination in TCR signaling and uncovers a novel role for LAT in driving T-cell differentiation.

ABSTRACT Pandemic influenza is a major public health concern, but conventional strain-matched vaccines are unavailable early in a pandemic. Candidate “universal” vaccines targeting the viral antigens nucleoprotein (NP) and matrix 2 (M2), which are conserved among all influenza A virus strains and subtypes, could be manufactured in advance for use at the onset of a pandemic. These vaccines do not prevent infection but can reduce disease severity, deaths, and virus titers in the respiratory tract. We hypothesized that such immunization may reduce virus transmission from vaccinated, infected animals. To investigate this hypothesis, we studied mouse models for direct-contact and airborne transmission of H1N1 and H3N2 influenza viruses. We established conditions under which virus transmission occurs and showed that transmission efficiency is determined in part at the level of host susceptibility to infection. Our findings indicate that virus transmission between mice has both airborne and direct-contact components. Finally, we demonstrated that immunization with recombinant adenovirus vectors expressing NP and M2 significantly reduced the transmission of virus to cohoused, unimmunized mice in comparison to controls. These findings have broad implications for the impact of conserved-antigen vaccines, not only in protecting the vaccinated individual but also in protecting others by limiting influenza virus transmission and potentially reducing the size of epidemics. IMPORTANCE Using a mouse model of influenza A virus transmission, we demonstrate that a candidate “universal” influenza vaccine both protects vaccinated animals from lethal infection and reduces the transmission of virus from vaccinated to nonvaccinated mice. This vaccine induces immunity against proteins conserved among all known influenza A virus strains and subtypes, so it could be used early in a pandemic before conventional strain-matched vaccines are available and could potentially reduce the spread of infection in the community.

Conventional influenza vaccines are designed to elicit antibodies to strainspecific antigens, leaving a public health gap when novel viruses break out unexpectedly.As examples, strain-matched vaccine became available too late in the pandemic of 2009 to protect against the fall wave, and drift viruses are sometimes divergent enough to cause vaccine failure (eg, A/Sydney in 1997).Available vaccines are also inadequate to protect against various zoonotic strains, including avian influenza A(H5N1), influenza A(H7N7), and most recently influenza A(H7N9).For this reason, there is much interest in developing vaccines based on conserved influenza virus features that can provide protection regardless of strain.These are usually designed for influenza A virus, but such vaccines can also be made for influenza B virus.Universal influenza vaccines

Emerging technologies result when advances and innovation in technology lead to discoveries. Often emerging technologies stimulate novel research in medical product development that contribute to new approaches to manufacturing and can improve the quality of products. By supporting investments in agency coordination, staff training and professional development, regulatory science research, stakeholder engagement, and enhancing opportunities for expert input, the U.S. Food and Drug Administration plays a critical role in translating innovations into novel safe and effective medical products that improve the public health.

Among approximately 1000 adenoviruses from chimpanzees and bonobos studied recently, the Pan Adenovirus type 3 (PanAd3, isolated from a bonobo, Pan paniscus) has one of the best profiles for a vaccine vector, combining potent transgene immunogenicity with minimal pre-existing immunity in the human population. In this study, we inserted into a replication defective PanAd3 a transgene expressing a fusion protein of conserved influenza antigens nucleoprotein (NP) and matrix 1 (M1). We then studied antibody and T cell responses as well as protection from challenge infection in a mouse model. A single intranasal administration of PanAd3-NPM1 vaccine induced strong antibody and T cell responses, and protected against high dose lethal influenza virus challenge. Thus PanAd3 is a promising candidate vector for vaccines, including universal influenza vaccines.