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Paper of the Month September 2011

DNA priming and influenza vaccine immunogenicity: two phase 1 open label randomised clinical trials



Julie E Ledgerwood*, Chih-Jen Wei*, Zonghui Hu, Ingelise J Gordon, Mary E Enama, Cynthia S Hendel, Patrick M McTamney, Melissa B Pearce, Hadi M Yassine, Jeffrey C Boyington, Robert Bailer, Terrence M Tumpey, Richard A Koup, John R Mascola, Gary J Nabel, Barney S Graham, and the VRC 306 Study Team



Because the general population is largely naive to H5N1 influenza, antibodies generated to H5 allow analysis of novel influenza vaccines independent of background immunity from previous infection. We assessed the safety and immunogenicity of DNA encoding H5 as a priming vaccine to improve antibody responses to inactivated influenza vaccination.


In VRC 306 and VRC 310, two sequentially enrolled phase 1, open-label, randomised clinical trials, healthy adults (age 18–60 years) were randomly assigned to receive intramuscular H5 DNA (4 mg) at day 0 or twice, at day 0 and week 4, followed by H5N1 monovalent inactivated vaccine (MIV; 90 μg) at 4 or 24 weeks, and compared with a two dose regimen of H5N1 MIV with either a 4 or 24 week interval. Antibody responses were assessed by haemagglutination inhibition (HAI), ELISA, neutralisation (ID80), and immunoassays for stem- directed antibodies. T cell responses were assessed by intracellular cytokine staining. After enrolment, investigators and individuals were not masked to group assignment. VRC 306 and VRC 310 are registered with ClinicalTrials.gov, numbers NCT00776711 and NCT01086657, respectively.
Findings In VRC 306, 60 individuals were randomly assigned to the four groups (15 in each) and 59 received the vaccinations. In VRC 310, of the 21 individuals enrolled, 20 received the vaccinations (nine received a two-dose regimen of H5N1 MIV and 11 received H5 DNA at day 0 followed by H5N1 MIV at week 24). H5 DNA priming was safe and enhanced H5-specific antibody titres following an H5N1 MIV boost, especially when the interval between DNA prime and MIV boost was extended to 24 weeks. In the two studies, DNA priming with a 24-week MIV boost interval induced protective HAI titres in 21 (81%) of 26 of individuals, with an increase in geometric mean titre (GMT) of more than four times that of individuals given the MIV-MIV regimen at 4 or 24 weeks (GMT 103–206 vs GMT 27–33). Additionally, neutralising antibodies directed to the conserved stem region of H5 were induced by this prime-boost regimen in several individuals. No vaccine-related serious adverse events were recorded.Interpretation DNA priming 24 weeks in advance of influenza vaccine boosting increased the magnitude of protective antibody responses (HAI) and in some cases induced haemagglutinin- stem-specific neutralising antibodies. A DNAMIV vaccine regimen could enhance the efficacy of H5 or other influenza vaccines and shows that anti-stem antibodies can be elicited by vaccination in man.

Also see a related commentary:Two is better than oneShan LuPublished Online October 4, 2011

DOI:10.1016/S1473- 3099(11)70256-0

In The Lancet Infectious Diseases, Julie Ledgerwood and colleagues reported on results from a phase 1 clinical trial showing that initial immunisation of individuals with a DNA vaccine expressing the haemagglutinin antigen of an avian source H5 subtype infl uenza virus, greatly improved the protective antibody responses elicited by a subsequent immunisation with the conventional inactivated influenza vacc
ine. By contrast, administration of two doses of the same inactivated influenza vaccine had much lower antibody responses than did the vaccine preceded by the DNA prime. Therefore, unmatched (heterologous
) prime-boost was more effective than matched (homologous) prime boost despite the same haemagglutinin antigen being used in both immunisation regimens, a finding gaining more attention lately.

Human beings are living under the threat of two types of influenza infections. One is seasonal influenza, which peaks in the winter seasons. Every year, the WHO issues guidelines on the selection of a formulation of a vaccine against seasonal influenza that covers the main circulating viruses during that period. The second type is pandemic influenza, which emerges suddenly and is transmitted quickly to a large proportion of the human population worldwide. Healthy people develop various degrees of immunity against seasonal influenza because of repeated natural exposure and, in some groups of people, through annual immunisations.

By contrast, the main threat of pandemic influenza is that people do not have preexisting immunity to minimise the effect of infection in public health. Timely development of protective immunity in the community is important to prevent the spread of an outbreak of pandemic influenza. A vaccine against pandemic influenza requires two doses to be effective, whereas the annual influenza vaccination against seasonal influenza only requires one inoculation. This distinction is a direct result of the differences in pre-existing immunity between these two types of influenza infections. Additionally, either higher dosing or the inclusion of an adjuvant is needed to achieve sufficient immunogenicity for vaccines against pandemic influenza that are currently available. Ledgerwood and colleagues validated a new strategy to improve the immunity of an influenza vaccine in naive human hosts, a strategy that had been reported in previous preclinical studies. Without changing the total number of immunisations, administration of a DNA vaccine is a more effective prime immunisationthan the inactivated influenza vaccine. This finding is especially important because a major limiting factor in the preparation against pandemic influenza is the restricted manufacturing capacity to produce enough doses of vaccines in a short period of time to cover the population in need.

If the vaccination strategy presented in this study was followed, the total amount of traditional vaccines against influenza would be reduced by half, which would allow more individuals to be vaccinated in a timely manner. The DNA vaccine is required as a second component with added complexity in this new prime-boost strategy. Although not shown in this report, preclinical studies have suggested that DNA primed antibody responses are usually long-lasting. Taking together, the report supports the idea of a prepandemic vaccination, since various DNA vaccines can easily be mixed together to provide broad coverage against several potential pandemic influenza viruses (even across different subtypes), long before any outbreak. Hosts primed with DNA vaccines are very likely to have reduced morbidity and mortality, even without a boost. This strategy gives public health officials the means needed to decrease the demand of traditional vaccines at the time of an outbreak of pandemic influenza. Moreover, this study1 is a milestone for DNA vaccine development. When used alone in man, DNA vaccines have not been sufficiently immunogenic, even with the use of various molecular adjuvants. By contrast, DNA vaccination is very effective in priming the human immune system to amplify the immune response when followed by a boost vaccination with either protein or viral vector vaccines. Ledgerwood and colleagues went further to show that DNA priming could be more effective than a type of influenza vaccine that has been licensed for human use. More studies are needed to see if doses of DNA and inactivated influenza vaccine can be reduced when such a heterologous prime-boost approach is used. It will also be interesting to compare the present approach with inactivated vaccines formulated with various adjuvants, particular results on the longevity of any recorded protective antibody responses. The results from the present study show that a long resting period between the prime and the boost is needed to achieve high protective antibody responses. Although the resting period between vaccinations is well known to be important in traditional vaccinology, such effect was remarkably clear in the present study.

This setting will offer a unique model to further dissect the mechanisms behind this phenomenon. By sequentially reducing the time between the prime and boost immunisations, it might be possible to determine whether antibody affinity maturation is implicated if the sequential sequence changes of immunoglobulin genes of protective antibodies are monitored. A well designed clinical study not only answers the clinical question at hand but also provides great insight to human biology. By using two vaccine components instead of one, Ledgerwood and colleagues certainly stimulated our thoughts more than the authors would have initially hoped.