Making a Candidate Vaccine Virus (CVV) for a HPAI (Bird Flu) Virus

Background

A candidate vaccine virus (CVV) is an influenza (flu) virus that has been prepared by CDC or another public health partner that can be used by vaccine manufacturers to produce a flu vaccine. In addition to preparing CVVs for seasonal flu vaccine production, CDC routinely develops CVVs for novel avian influenza (bird flu) viruses with pandemic potential as part of pandemic preparedness activities. Some novel bird flu viruses with pandemic potential are "highly pathogenic avian influenza" (HPAI) viruses, which means they are deadly to domestic poultry, including chickens. Data collected through global and animal flu surveillance informs the selection of CVVs, and experts choose CVVs against bird flu viruses in nature ("wild type" viruses) that pose a risk to human health.

The creation of a CVV for a novel bird flu virus is a multistep process that takes months, from start to finish. Creating a bird flu CVV is usually more complicated than the process for creating a seasonal flu CVV. There are five steps involved in the creation of a bird flu CVV, including the following:

1. Production of the candidate vaccine virus (CVV) ;
2. Quality assessment;
3. Determination of attenuation;
4. Request for USDA Select Agent exclusion; and
5. Distribution of the CVV to vaccine manufacturers and other stakeholders.

These steps are described below.

Production of the candidate vaccine virus (CVV)

Flu vaccines protect against specific flu viruses, so the first step in creating a CVV against a particular bird flu virus is to identify the wild type bird flu virus that is posing or may pose a risk to human health.

Two qualities are required of a CVV: First, the CVV must be capable of stimulating a protective immune response against that wild type bird flu virus. Second, the CVV must grow well in eggs. Most flu vaccines today are produced using egg-based technology, which requires that the CVVs be grown in chicken eggs so the CVV needs to grow well in chicken eggs. (For more information about how vaccines are made, see "How Influenza (Flu) Vaccines Are Made.") Highly pathogenic bird flu viruses cause severe illness and death in birds and destroy chicken eggs, and are therefore very difficult to grow in eggs. In addition, HPAI viruses cause severe illness and death in animal models of human disease. Therefore, the wild type virus found in nature cannot be used to make the CVV because they won't grow in eggs and might be dangerous to people. Scientists at CDC use "reverse genetics" to create an attenuated (i.e., weakened or milder) form of the bird flu virus that will not cause severe illness in birds (so as to not pose a threat to agricultural interests) and that also will grow well in chicken eggs (so that vaccine manufacturers can use it to produce vaccine).

Using reverse genetics, CDC scientists take two genes from the bird flu virus — the neuraminidase (NA) and a modified form of the hemagglutinin (HA) gene — and they combine those two genes with six genes of a commonly used human flu virus that grows well in chicken eggs. The HA and NA from the bird flu virus are either prepared synthetically or by using the viral RNA of the wild type virus, if it is available. The HA is modified by removing the site that is responsible for making the virus deadly to birds and chicken eggs, called the "polybasic cleavage site." The prepared DNA is introduced into cells (typically "Vero" cells in the laboratory) and allowed to grow for three days. Then it is collected from the cells and injected into eggs to grow virus stocks. The virus stocks undergo testing to determine if a sufficient amount of virus is present in the eggs, and the CVV is analyzed to determine if it is suitable for vaccine development in terms of compliance with regulatory requirements.

A CVV must be made according to FDA "good laboratory practice" (GLP) regulations for biologics for human use. GLP is a quality system concerned with the organizational process and the conditions under which vaccine development is planned, performed, monitored, recorded, archived and reported per FDA guidelines.

Ideally, the attenuated virus resulting at the end of this process is a virus that is antigenically "like" the wild-type bird flu virus. This virus should stimulate the desired immune response and grow well in chicken eggs.

Quality Assessment

Once the CVV has been created using reverse genetics and grown in eggs (i.e., rescued), the CVV undergoes a number of tests as part of quality assessment. These steps are listed and described below.

• Gene Sequence Verification: Flu experts verify that the CVV produced is genetically stable and hasn't undergone any unintended changes or mutations. PCR and full genome sequencing are used to ensure the CVV is as close as possible to the wild type bird flu virus it is intended to protect against.
• Impurity Testing: Flu experts check to make sure that the CVV does not contain bacteria or fungi.
• Exclusivity Testing: The CVV is tested to ensure that it only contains the virus intended and is not mixed with other viruses.
• Trypsin Dependent Plaque Assay Testing: The "trypsin dependent plaque assay" is a laboratory test that ensures that the polybasic cleavage site (i.e., the part of the virus that makes the virus deadly in poultry and chicken eggs) has been removed.
• Embryo Lethality Testing: This test confirms that the CVV does not kill chicken embryos. This is a required for egg-based vaccine production.
• Antigenic Characterization: CDC uses the hemagglutinin inhibition (HI) test to antigenically characterize the CVV to ensure that the CVV is antigenically similar to the bird flu virus in nature that the CVV is intended to protect against. Antigenic similarity in this instance means antibodies produced in response to the CVV also will recognize and target the corresponding wild-type bird flu virus.

Determination of Attenuation

Following quality assurance tests, the CVV goes through a series of animal tests intended to confirm that the resulting virus stock is attenuated (i.e., made milder or weakened). Determination of attenuation requires testing in chickens and ferrets. Specific steps are described below:

• Pathogenicity Testing in Chickens: Chickens are inoculated with the CVV and observed to make sure they don't get sick or die. This testing verifies that the CVV is not highly pathogenic in domestic poultry. This means the CVV will not harm agricultural interests and can be safely used during egg-based production. Pathogenicity testing in chickens is done by an outside collaborator, such as the United States Department of Agriculture's (USDA's) Southeastern Poultry Research Laboratory (SEPRL). The results of this testing are then shared with CDC.
• Pathogenicity testing in ferrets: Ferrets also are inoculated with the CVV and observed to ensure the CVV does not cause severe illness. Flu viruses infect and cause disease in ferrets in a way that is similar to humans, and so ferrets often are used as a model to infer flu disease and transmission characteristics in people.

This is the final step to ensure that the CVV has been properly attenuated and is safe for birds and mammals.

Request for CVV Exclusion from the USDA Select Agents List

Next, a request is filed with the USDA to get the CVV excluded from the "Select Agent" list. Certain biological agents or toxins that pose a potential severe threat to public health and safety are designated as "Select Agents." (For more information on select agents, see the Federal Select Agent Program website.) Highly pathogenic bird flu viruses are Select Agents, and so a CVV made from one of these viruses is treated as a Select Agent until an exclusion is granted by USDA. This is necessary before the CVV can be given to flu vaccine manufacturers. To obtain USDA Select Agent exclusion, CDC prepares and submits appropriate documentation to USDA, including all of the virus attenuation verification data. USDA then reviews the application and decides whether the CVV can be excluded as a Select Agent.

Distribution to Vaccine Manufacturers and Other Stakeholders

The final step is to ship the CVV. The CVV is shared with other World Health Organization (WHO) Collaborating Centers, Essential Regulatory Laboratories (ERLs) and vaccine manufacturers.