Rationale Vaccine Design For Canine H3N8 Influenza Biology Essay

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Influenza virus is a type of RNA virus that cases influenza in human and animals. It is genus of the Orthomyxoviridae family in virus taxonomy. Influenza virus can infect human, swine, equine, avian and many other kind of animal species. It can be divided into three subtypes (influenza A, B, and C virus) depends on the antigenic differences in two of their internal proteins, nucleoprotein and matrix protein.

Influenza A virus is a kind of virus that undergoes antigenic change from frequently and cause regional influenza pandemic in human. The surface antigen of the virus is likely to change (drift) and forms a new subtype of influenza A virus.

Influenza A viruses are negative-sense, single-stranded RNA viruses containing eight viral RNA segments, with an envelope covered outside. The influenza A virus can be categorized into several subtypes based on its two protein components on the surface of viral envelope: hemagglutinin (HA) and neuraminidase (NA). There are 17 subtypes of hemagglutinin (H1~H17), and 9 subtypes of neuraminidase (N1~N9). Theoretically, there are 153 (19*7) possible combinations of H and N, therefore there are 153 subtypes of influenza A virus. Among all the subtypes, H1N1, H2N2, and H3N2 are mainly infectious to human. H5, H7, and H9 strains are especially dangerous to birds. Many other subtypes have their natural hosts vary from birds to animals. Normally, influenza A virus can cause acute upper respiratory tract infection, and can be transmitted through air rapidly.

Fig. 1. Protein and RNA composition of Influenza A virus.

HA, haemagglutinin;

NA, neuraminidase;

M1, M2, matrix proteins;

NP, nucleoprotein;

NS, non-structural proteins;

PA, PB1, PB2, proteins involved in virus replication.

The H3N8 subtype of influenza A virus has been isolated from horses in 1963 (Waddell et al., 1963), and this virus still circulates in many horses around the world today. Equine influenza virus (EIV) of the family Orthomyxoviridae is a major cause of equine respiratory disease.

In 2004, 22 hound dogs were infected with respiratory disease and had the symptom of fever and cough. 14 dogs were recovered from the illness in the following 10 to14 days, but 8 dogs died acutely with hemorrhage in the respiratory tract (Crawford et al., 2005). Later, the canine influenza virus (CIV) subtype was isolated from the dead dog bodies. Sequence comparison with known influenza virus genes and phylogenetic analyses indicated that, the canine isolate, named A/canine/Florida/43/2004 (canine/FL/ 04), as an influenza A H3N8 virus, was homologous to contemporary equine influenza viruses, and they shared >96% sequence identity (Crawford et al., 2005).

In 2002, another case was reported in the United Kingdom on the occurrence of CIV H3N8-associated respiratory disease (Daly et al., 2008). From January to May 2005, respiratory disease broke out in 11 states of US (Florida, Texas, Arkansas, Arizona, West Virginia, Kansas, Iowa, Colorado, Rhode Island, Wisconsin, and Massachusetts) and attacked a population of ~20,000 greyhounds. Sequence analysis on the virus isolated from the dead dogs identified a close relationship to H3N3 influenza A virus. These data indicated sustained circulation of a canine/FL/04-like virus in this population (Crawford et al., 2005).

The high prevalence of canine influenza infection in greyhound may suggest a high risk of infection in pet dogs. However, when a dog is infected with CIV, the dog owner may not realize that the dog is sick until the dog begins coughing and has greenish nasal discharge. The onset of coughing means that the dog is likely to develop pneumonia. It normally takes a few days before the dog finally get medical care, during which the virus is able to magnified in the host and induce lesion in lung and respiratory tract. Furthermore, experiment results showed that CIV spreads rapidly from infected dogs to other susceptible dogs though direct contact (Faris F. Jirjis et al,. 2010). Since CIV also have been isolated from several breeds of dogs in the United States in some cases (Payungporn et al., 2008; Animal Health Diagnostic

Center), all breeds of dogs are susceptible to CIV infection (Dubovi and Njaa, 2008)

Due to the lack of either natural or acquired immunity in dogs, all breeds of dogs have the risk of suffering from CIV H3N8 infection. There is also a possibility that a dog may die of the acute disease induced by CIV H3N8 infection, and it would be too late for the dog owner to safe the dog by that time. Therefore, it is necessary for dog owners to give their dogs vaccination to help prevent infection from dog to dog and to keep dogs healthy even during CIV outbreaks. 在写一些市场分析

In this report, we proposed a new vaccine designed to effectively prevent canine H3N8 influenza A virus infection and transmission in dogs.

Product Description

Our CIV H3N8 Vaccine is a live freeze-dried vaccine derived from attenuated strain of CIV H3N8 influenza A virus, and can be vaccinated to dogs by oral. So you don't have to worry about whether your dog will suffer from an uncomfortable injection shot. Just let your lovely dog have some drops on the nose and it is vaccinated!

The vaccine should be used for dogs of 6 months old age and older. The product meets the requirements of WHO when tested (I wish).


This vaccine are aimed at preventing canine H3N8 influenza infection in dogs of 6 months old age and older.


Live attenuated CIV H3N8 virus.


Our CIV H3N8 vaccines are derived from CIV H3N8 viruses which were isolated from canines infected by CIV H3N8 influenza A virus previously. CIV H3N8 viruses are existed in the nasal discharge of infected canines.

Antigen titers -原浓度 10-fold serially diluted 十倍梯度稀释犬副流感弱毒(CPIV/A-20/8æ ª)≥104.5TCID50/0.1mlï¼›


Dilute the vaccine with distilled water before usage. Apply immediately after dilution.

Dosage and Administration

The vaccine is intended to be administered intranasally via a spray device, 0.20 ml in each nostril.

This vaccine should be given a second time after 3~4 weeks of first vaccination. We suggest a frequency of annual vaccination after the two initial doses.

Contraindications and Precautions

Do not give this vaccination to dogs that are immunodeficient, pregnant, or already having an influenza infection.

Do not use anti-virus drugs and interferons during the 4 days before and after the CIV vaccination.

Do not vaccinate the dog with any other kind of live attenuated virus vaccine in the following 7 days of the CIV vaccination.

Drug Interaction and Other Interactions

The efficacy of this CIV vaccination may be reduced by antivirus drug, and the live-attenuated CIV H3N8 virus compositions in the vaccine product are also likely to have an interaction with other kind of influenza virus that affect canine.


This vaccine could conserve its validity for two years if stored in dark and frozen between -10℃ to -20℃.

Adverse Events

Might include local pain and myalgia (pain in muscle), other adverse events are not known yet.

How the vaccine works (immunological reasoning)

The immune response to our CIV H3N8 live attenuated influenza vaccine (LAIV)

Viruses are intracellular microorganisms, and they have to survive and replicate within living cells. Influenza viruses typically infect a wide variety of cell population by using normal cell surface molecules as receptor to enter the cell. After entering cells, viruses often use the existing machinery of the host cells to produce their viral proteins and nucleic acids for viral replication. Viral replication interferes with normal cell protein synthesis and functions, thus lead to injury and finally the death of infected cell, and moreover, tissue injury and disease occur result from the accumulated cell infection in the host. The host cells are lysed and viruses are released to infect other cells. Innate and adaptive immune response will be triggered to block infection and eliminate infected cells.

During H3N8 influenza virus infection, the viral protein and nucleic acids in cellular counterparts are first distinguished by pattern recognition receptors (PRR). The single-stranded RNAs with a 5'-triphophate inside the influenza A virus H3N8 are normally not found in the cytoplasm of uninfected canine cells, thus they will be detected by cytoplasmic protein RIG-I as foreign antigens. The RIG-I will bind these viral RNAs and onset a series of innate immune reactions, including cytokine synthesis. The toll-like receptors (TLR7/8) also recognize the single-stand RNAs from the virus through receptor-associate kinases, and stimulate the production of microbicidal substances and cytokines in the phagocytes. When recognized by the TLR in neutrophils and macrophages, the viruses will be ingested into particular intracellular vesicles (lysosomes and phagolysosomes). Neutrophils and macrophages also express some receptors that activate the cells to produce cytokines and microbicidal substances and receptors to stimulate the cells migrating to infection cites.

The infection of the host cells by H3N8 viruses inhibits the expression of class I MHC, therefore the ligands for inhibiting NK cell receptors are lost. The NK cells are activated from their normal state of inhibition, and ligands for activating receptors are expressed. NK cells kill the virus-infected cells and those cells that stop expressing class I MHC molecules. NK cells respond to IL-12 produced by macrophages, and they secret cytokines, mainly IFN-γ,which activates the macrophages to kill phagocytosed microbes.

Immature dendritic cells express Toll-like receptors in the membranes, and use this receptors to capture H3N8-infected cells as antigens and to endocytose and break down these antigens, process the proteins into peptides capable of binding MHC molecules. Cytokines produced by innate immune reactions bind to microbes, and the cytokine-microbe combination activates the dendritic cells. The dendritic cells stop adhering to the epithelia and began to transport viral antigens to lymph nodes. During this migration, the immature dendritic cells mature and are able to display antigens to naïve T cells and activate the cells.

After naïve T cells mature, they proliferate mainly in response to the autocrine growth factor IL-2. Naïve CD4+ T cells differentiate into subsets of effector cells (for example, TÓŠ1 and TÓŠ2 cells) that produce different sets of cytokines(for example, IFN-γ, IL-4 and IL-5), and therefore perform different effector functions. Naïve CD8+ T cells differentiated into CTLs, which are effector T cells migrating to infection sites. The CTLs recognize and kill target cells expressing influenza viral peptide antigens in association with class I MHC molecule. Moreover, the CD8+ CTLs activate the phagocytes, which kill phagocytosed microbe at infection sites.

Helper T cells (i.e. CD4+ T cells) then activate specific B cells through a phenomenon known as an Immunological synapse. Activated B cells subsequently produce antibodies which assist in inhibiting pathogens until phagocytes (i.e. macrophages, neutrophils) or the complement system for example clears the host of the pathogen(s). With a T-dependent antigen, the first signal comes from antigen cross linking the B cell receptor (BCR) and the second signal comes from co-stimulation provided by a T cell.

Helper T cells (CD4+ ) then activate B lymphocytes through immunological synapse, to help B cells produce antibodies. The hemagglutinin in the H3N8 influenza A virus is the main determinant of virulence, and plays an crucial role in the virus life cycle. During the immune response to the H3N8 infection, antibodies to the viral surface glycoprotein hemagglutinin and neuraminidase are produced to work against the infection. NK cells recognize antibody-coated targets by FcγRIIIa (CD16), and kill targeted cells. Nevertheless, antibodies to the conserved internal antigens, matrix proteins and nucleoproteins are not helpful. The cytotoxic T-cells (CTL) respond directly against matrix proteins and nucleoproteins. Though this response does not offer protection, it is important for cleaning the virus and recovering from the illness.

When vaccinating though nostrils, the mucosal tissues are the main entry of LAIV. The mucosal immune system provides the first defense against virus infection. Nasal secretions contain specific antibodies to neutralize hemagglutinin (HA) and neuraminidase (NA). These antibodies are mainly IgA and are produced in local. IgG and IgM are also produced specifically to HA.

The mucosal immunity will response to the CIV H3N8 LAIV, and secret antibodies in the upper respiratory tract. IgA and IgM are the major antibodies in mucosa that neutralizing H3N8 and preventing pathogen entry. Immunoglobulin A (IgA) is secreted to protect the upper respiratory tract, and serum IgG is responsible for protection of lower respiratory tract. IgA and IgM will function intracellularly to inhibit replication of virus. Locally, nasal mucosa will secret IgA to neutralize influenza HA and NA.


Advantage of H3N8 Live Attenuated Influenza Vaccine (LAIV)

The goal of LAIV is to induce a secretory and systemic immune response which most similar to the immune response triggered by nature infection. Live attenuated influenza virus vaccine is expected to replicate inside the host body, thus it mimics the nature infection of influenza virus, which may lead to long-lasting immunity to infecting virus. LAIV vaccine stimulates both local and system immunity. However, there is also a risk of unexpected enhancement of infection given by the live attenuated virus vaccine.

This H3N8 LAIV doesn't need any adjuvant. Small dose of LAIV can induce a quick and strong response from canine immune system by only few times (2 times) of vaccination. The production of LAIV could have a relatively low cost and high yield.

Storage and Transportation

LAIV should be stored and transported in dark at a very low temperature. Thus the cost increased by the specific storage and transportation condition.

Because influenza virus undergoes unpredictable changes frequently, the effective protection provided by the host's immunity would only last for a period (may be a few years) before the new strains of influenza virus start circulating.

http://onlinelibrary.wiley.com/doi/10.1111/j.0300-9475.2004.01382.x/full Influenza Virus: Immunity and Vaccination Strategies. Comparison of the Immune Response to Inactivated and Live, Attenuated Influenza Vaccines