More antibody with less antigen: can immunogenicity of attenuated live virus vaccines be improved?

Abstract

New or improved vaccines against viruses such as influenza, parainfluenza types 1-3, measles, dengue, and respiratory syncytial virus would prevent an enormous burden of morbidity and mortality. Vaccines or vaccine candidates exist against these viral diseases, but all could potentially be improved if the immunogenicity of the vaccine could be enhanced. We found that the immunogenicity in primates of a live-attenuated vaccine candidate for parainfluenza virus type 3, an enveloped RNA virus that is an important etiologic agent of pediatric respiratory tract disease, could be enhanced by expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) from an extra gene inserted into the genome of a cDNA-derived virus. Expression of GM-CSF by the live attenuated recombinant virus did not per se affect the level of pulmonary viral replication in rhesus monkeys after topical administration, which was 40-fold lower than that of WT parainfluenza virus type 3. Despite that, the expressed extra gene augmented the virus-specific serum antibody response to a level that was (i) 3- to 6-fold higher than that induced by the same virus with an unrelated RNA insert of equal length and (ii) equal to the response induced by nonattenuated WT virus. In addition, topical immunization with the attenuated virus expressing GM-CSF induced a greater number of virus-specific IFN-gamma-secreting T lymphocytes in the peripheral blood of monkeys than did immunization with the control virus bearing an unrelated RNA insert. These findings show that the immunogenicity of a live-attenuated vaccine virus in primates can be enhanced without increasing the level of virus replication. Thus, it might be possible to develop live-attenuated vaccines that are as immunogenic as parental WT virus or, possibly, even more so.

Fig 1.

Schematic representation of the RNA genomes of B/HPIV3/GM-CSF and B/HPIV3/CAT. BPIV3 genes are represented by white bars, HPIV3 by gray bars, and the inserted genes (human GM-CSF or bacterial CAT sequence in reverse orientation) by hatched bars. Gene-start and gene-end sequences are indicated by small black bars upstream and downstream, respectively, of each gene. The viral gene designations are as follows: N, nucleocapsid protein; P, phosphoprotein; M, internal virion matrix protein; F, fusion glycoprotein; HN, hemagglutinin-neuraminidase glycoprotein; L, polymerase protein. Extragenic leader (Le) and trailer (Tr) sequences are depicted at 3′ and 5′ ends of the genomic RNAs.

Fig 2.

Reciprocal HPIV3-specific serum antibody titers determined by HAI assay for monkeys immunized with the indicated virus. Eight monkeys per group were immunized with B/HPIV3/GM-CSF, B/HPIV3/CAT, or WT HPIV3. Dots represent the serum HAI antibody titers for each monkey on days 28 and 56; bars represent the means. Before immunization, all animals were confirmed to be negative for HPIV3-specific serum antibodies (HAI titers were <1:4).

Fig 3.

Numbers of peripheral blood T lymphocytes isolated after immunization with B/HPIV3/GM-CSF (group A) or B/HPIV3/CAT (group B) that secrete IFN-γ in response to specific in vitro stimulation with B/HPIV3. The number of IFN-γ-secreting T lymphocytes per 400,000 cells was determined by enzyme-linked immunospot. Individual monkeys are indicated by dots, and means are shown by bars.