Adenoviral Vectors as Vaccines for Emerging Avian Influenza Viruses

Abstract

It is evident that the emergence of infectious diseases, which have the potential for spillover from animal reservoirs, pose an ongoing threat to global health. Zoonotic transmission events have increased in frequency in recent decades due to changes in human behavior, including increased international travel, the wildlife trade, deforestation, and the intensification of farming practices to meet demand for meat consumption. Influenza A viruses (IAV) possess a number of features which make them a pandemic threat and a major concern for human health. Their segmented genome and error-prone process of replication can lead to the emergence of novel reassortant viruses, for which the human population are immunologically naïve. In addition, the ability for IAVs to infect aquatic birds and domestic animals, as well as humans, increases the likelihood for reassortment and the subsequent emergence of novel viruses. Sporadic spillover events in the past few decades have resulted in human infections with highly pathogenic avian influenza (HPAI) viruses, with high mortality. The application of conventional vaccine platforms used for the prevention of seasonal influenza viruses, such as inactivated influenza vaccines (IIVs) or live-attenuated influenza vaccines (LAIVs), in the development of vaccines for HPAI viruses is fraught with challenges. These issues are associated with manufacturing under enhanced biosafety containment, and difficulties in propagating HPAI viruses in embryonated eggs, due to their propensity for lethality in eggs. Overcoming manufacturing hurdles through the use of safer backbones, such as low pathogenicity avian influenza viruses (LPAI), can also be a challenge if incompatible with master strain viruses. Non-replicating adenoviral (Ad) vectors offer a number of advantages for the development of vaccines against HPAI viruses. Their genome is stable and permits the insertion of HPAI virus antigens (Ag), which are expressed in vivo following vaccination. Therefore, their manufacture does not require enhanced biosafety facilities or procedures and is egg-independent. Importantly, Ad vaccines have an exemplary safety and immunogenicity profile in numerous human clinical trials, and can be thermostabilized for stockpiling and pandemic preparedness. This review will discuss the status of Ad-based vaccines designed to protect against avian influenza viruses with pandemic potential.

Keywords: adenoviral vector; adenovirus; avian influenza; highly pathogenic; highly pathogenic avian influenza; immunogenicity; influenza; vaccine.

Figure 1

Schematic Diagram Showing Zoonotic Cycle of Influenza Viruses. Influenza A viruses can infect multiple animal species, which increases the probability of cross-species transmission events. Migratory and aquatic birds represent natural reservoirs for avian influenza viruses, and pigs act as a mixing vessel, allowing the reassortment of diverse influenza viruses. The process of reassortment could lead to the emergence of novel influenza subtypes which are better adapted for infection and transmission in humans. Several barriers to this process also exist, including, but not limited to receptor usage preferences. Direct infection of humans with avian influenza viruses is an infrequent event. However, the potential for adaptation while maintaining high pathogenicity is a major concern and drives efforts to develop improved vaccines against emerging avian influenza viruses. Figure created with ^©BioRender - Biorender.com.

Figure 2

Schematic Diagram of IAV Structure and Reassortment. (A) Figure shows a schematic cross-section of the influenza virus virion with main components labeled. Surface glycoproteins, trimeric hemagglutinin (HA) and tetrameric neuraminidase (NA), play important role in viral entry and egress and are major targets for immune responses following infection or immunization. In particular, the highly conserved stalk domain of HA is a target for universal influenza virus vaccines. Note: HA stalk and NA stalk are not shown as trimeric or tetrameric structures. Internal, highly conserved antigens matrix protein-1 (M1) and nucleoprotein (NP) are targets for cytotoxic T lymphocytes (CTLs). Note: icons for NP, which coats the viral RNA, and the viral ribonucleoproteins (vRNPs) which contain viral RNA, NP and polymerase are not shown. (B) Influenza A viruses (IAVs) can evolve to generate viruses with pandemic potential by antigenic shift, using a process of genome reassortment. Co-infection of susceptible cells with more than one distinct IAV can result in the selection of progeny with shuffled gene segments and potentially a new HA or NA, against which humans have no prior immunity. Figure created with ^©BioRender - Biorender.com.

Figure 3

Schematic of Influenza Virus Life Cycle and Targets for Protective Antibodies. The life cycle of influenza viruses has several major steps in which inhibition by neutralizing or protective antibodies can occur. (1) Viral entry in the respiratory tract is facilitated by the enzymatic activity of the viral neuraminidase (NA), which cleaves mucins to allow access to respiratory cells. Anti-NA antibodies, or anti-hemagglutinin (HA) antibodies which block the enzymatic function of NA could potentially inhibit this process. (2) Viral entry is mediated by binding of the head of HA to sialic acid receptors on the surface of cells, followed by endosomal escape by fusion of the viral and endosomal membrane. Antibodies which bind to the HA head domain can block this interaction and can confer sterilizing protection from infection. (3) Alternatively, neutralizing antibodies against HA can block the post-binding internalization of influenza virus, or (4) its’ ability to fuse and escape from the endosome. (5) Viral ribonucleoproteins (vRNPs) are imported into the nucleus for viral transcription and replication. (6) mRNAs exported to the cytoplasm for translation. (7) HA and NA are trafficked to the Golgi for post-translational modification and subsequent presentation on the cell surface. Selected proteins return to the nucleus to participate in viral replication. Progeny vRNPs are exported out of the nucleus towards the plasma membrane for subsequent assembly and virion formation. (8) Anti-HA stalk antibodies can recognize HA on the surface of infected cells and engage Fc-mediated effector functions such as antibody-dependent cellular cytotoxicity, targeting the infected cell for degradation. (9) Viral packaging, assembly and egress takes place at the plasma membrane. This process can also be a target for anti-HA or anti-NA antibodies, which block egress. Anti-NA antibodies can do this by preventing new virions from being released from the surface of infected cells, or by the absence of NA activity causing new virions to aggregate. Figure is adapted from Krammer, 2019 (4). Note: icons are not to scale. HA stalk is trimeric (not shown) and NA stalk is tetrameric (not shown). Figure created with ^©BioRender - Biorender.com.

Figure 4

Approaches for Influenza Vaccine Development. Left panel: A schematic overview of conventional influenza virus vaccine platforms, including the live attenuated vaccine (LAIV), the split virion inactivated influenza vaccine (IIV) or IIV sub-virion vaccine, which has HA>NA content. Right panel: Newer vaccines being developed include recombinant HA protein, virus-like-particles or nucleic acid-based vaccines such as DNA or mRNA platforms. Center panel: Schematic overview of how non-replicating adenoviral (Ad) vectored vaccines work. DNA sequence encoding an influenza virus antigen is inserted into the dsDNA genome of the Ad vector under the control of a powerful promoter to drive expression. Once immunized, the DNA sequence coding for the influenza antigen is transcribed into mRNA and translated into protein which is expressed inside the host cells at the site of injection and/or within draining lymph nodes. This results in a robust CD8⁺ T cell response, as well as humoral immune responses directed towards the encoded transgene antigen. Note: the trimeric stalk of HA, or tetrameric stalk of NA are not shown in the diagram and icons are not to scale. Figure created with ^©BioRender - Biorender.com.

Figure 5

Strategies to Re-focus Humoral Immunity to the HA stalk. (A) Schematic diagram showing the substitution of the HA head domain to make chimeric HA (cHA) immunogens. The concept behind this approach is to graft an exotic HA head, for which humans have no prior immunity, to the stalk of a HA subtype which is common in humans (ie. H1 or H3). Sequential immunization with cHA immunogens in which the exotic head is swapped with each boost can re-focus humoral immunity to the conserved HA stalk. Note: Structures are schematic and do not represent authentic junctions for substitution of the HA head region. (B) Mosaic HA (mHA) design is conceptually similar to cHAs but only the major antigenic sites in the HA head domain are swapped for comparable regions in an exotic HA. This can be used as an alternative approach to re-focus antibodies towards the HA stalk domain, with the added benefit of retaining possible conserved epitopes in the HA head. mHA structures kindly provided by Dr. Felix Broecker and Prof. Peter Palese, ISMMS. (C) Structurally stabilized headless HAs have been engineered which completely lack the immunodominant HA head domain, allowing boosting of immune responses towards the stalk only. HA structures in (C) are reproduced with permission from Impagliazzo et al. (147). Reprinted with permission from AAAS (License 4907650635299). Figure created with ^©BioRender - Biorender.com.