Key Point

Using reverse genetics, researchers created a bivalent, attenuated live vaccine that protected mice against lethal challenge with both influenza and parainfluenza viruses. This technology could be used to develop vaccines active against several different viruses in a single dose.

NEW YORK CITY—Both influenza and parainfluenza viruses are among the most common causes of respiratory disease worldwide. Each year, influenza accounts for three million to five million cases of severe illness, while an estimated 16,300 to 96,500 persons are hospitalized because of human parainfluenza virus infections.

Although inactivated vaccines are effective at preventing influenza-related hospitalizations and deaths, the immune response is short-lived, necessitating annual vaccination against viral strains selected for the coming flu season. In contrast, live attenuated vaccines elicit long-lived, robust humoral and cellular responses. Unfortunately, for human parainfluenza viruses, no vaccines are available.

Recently, using a mouse model, researchers in the United States and Japan have developed a bivalent vaccine against both influenza and parainfluenza viruses.¹ They employed influenza A virus as a vaccine vector because “it elicits strong humoral and cellular responses, its genome is amenable to genetic modification, and the availability of 15 hemagglutinin (HA) and nine neuraminidase (NA) subtypes (HA and NA are the major viral antigens) would allow repeated immunization.”

REVERSE GENETICS USED IN VACCINE’S CREATION

The researchers set out to create a bivalent live vaccine that would provide protection not only against influenza but also parainfluenza virus infections. Employing reverse genetics, they generated an influenza A virus for which the ectodomain of the NA was replaced by that of the HN—a parainfluenza virus protective antigen.

Influenza A/WSN/33 (H1N1) virus (WSN) was grown in chicken eggs, as was murine parainfluenza virus type 1 Sendai virus (SeV). From these, a virus expressing both SeV and HN proteins (FluH/SeVHN) was generated. In vitro, the recombinant virus produced high titers and grew well, producing only one mutation after being passed 10 times through chicken eggs.

The pathogenicity of FluH/SeVHN was tested by intranasal infection of 4-week-old BALB/c mice (10^5.3, 10^6.3, or 10^7.3 egg infectious doses [EID₅₀] per mouse). Survival times and body weights were monitored for 14 days postinfection. Three days after infection, a subgroup of mice was killed, and nasal and lung viral titers were evaluated. To determine the dose lethal to 50% of mice infected with wild-type WSN or SeV, mice were infected intranasally with tenfold serial dilutions of virus and then observed for 14 days.

PUTTING THE VACCINE TO THE TEST

The efficacy of FluH/SeVHN vaccine was tested by challenging mice with lethal doses of wild-type viruses. A control group was given either an inactivated virus or saline solution. Twenty-seven days later, serum samples and trachea-lung and nasal washes were collected from a subset of mice and examined for virus-specific immunoglobulin A (IgA) or IgG antibodies. The remaining mice were challenged intranasally with a lethal dose of wild-type WSN or SeV and monitored daily for 28 days thereafter.

Compared with the control group, mice inoculated with the FluH/SeVHN virus at either 10^5.3, 10^6.3, or 10^7.3 EID₅₀ had elevated antibodies to SeV. Antibody titers to WSN—both IgA and IgG—were significantly elevated in all inoculated mice, but not in controls. For mice that were inoculated using the 10^5.3 EID₅₀ dose of the vaccine, there was no significant change in SeV titers. Thus, the ideal vaccine dose needed to produce a significant antibody response was a minimum of 10^6.3 EID₅₀.

When challenged with wild-type SeV or WSN, the inoculated mice were completely protected. Mice in the control group died six to 10 days postchallenge.

“FluH/SeVHN protected mice against lethal challenge with influenza and murine parainfluenza viruses. It also replicated efficiently in eggs, stably expressed the HN antigen, was highly attenuated in mice, and induced protective antibodies against both parainfluenza and influenza viruses in these animals. Hence, it meets the basic requirements for a live attenuated vaccine,” wrote the study authors.

One problem that can occur when viruses are used as vectors is that the immune response against the vector components may prohibit the repeated use of a single vector. By using influenza A (H1N1) as a vector, the authors pointed out, “the immunogenicity of the influenza virus vector is considered beneficial, since it would elicit immune responses against a societally and economically important pathogen.”

—Gale Jurasek

Reference
1. Maeda Y, Hatta M, Takada A, et al. Live bivalent vaccine for parainfluenza and influenza virus infections. J Virol. 2005;79:6674-6679.

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