Cellular immunity was determined by lymphocyte proliferation and cytokine production assays using peripheral blood mononuclear cells from vaccinated animals stimulated in vitro with WNE. Cell-mediated immune responses varied from animal to animal within each group. About half of the animals responded with lymphoproliferation, cytokine production, or both. Again, there was little difference in response between animals immunized with a 1- or microg dose of WNE in the vaccine formulations.
The benefit of suppressing revertant frequency in the inoculum is evident in Fig 7 : the magnitude of immunity to the vaccine increases by orders of magnitude as the initial frequency of the revertant is decreased.
The effect is strongest at low inoculum levels, pointing to the other solution—increase inoculum size. Small inocula that contain vaccine plus revertant are more prone to reduced immunity levels than are large inocula with little revertant.
Composition of the vaccine has the larger effect for the inoculm sizes and initial revertant fractions shown, as indicated by the contours being more horizontal than vertical. Smaller c values would lead to higher vaccine and immunity levels across the graphs.
Intuition also suggests that the deleterious effects of evolution can be reduced by increasing the inoculum size, provided the composition does not change: to achieve a threshold antigen level, a large inoculum requires less growth than a small one. Less growth reduces the potential for evolution—in the extreme, a large enough inoculum requires no vaccine growth, as with killed vaccines.
These conjectures are supported by Fig 7 : when the revertant frequency in the inoculum is high, increasing the inoculum size appreciably increases the magnitude of immunity; a much reduced benefit is seen when revertant frequency is low, likely because there is less evolutionary interference from the revertant.
These results suggest parallel benefits from reducing the frequency of the revertant in the inoculum and increasing the dose. Consideration of the gains from each could help choose an economically feasible strategy, since both purifying the inoculum and increasing its dose are likely to incur financial costs.
Whether and how well controlling the inoculum will work in practice will depend on details. Solutions may be quantitative rather than absolute. Intuition is useful for guidance but needs to be confirmed by formal analyses, guided by data from the specific implementation. Any live viral vaccine may evolve within the host. The potential for attenuated viruses to revert to wild-type virulence is well appreciated [ 1 , 2 ], even if it presents a problem for relatively few vaccines e.
There is also a potential for live, recombinant vector vaccines to evolve—our focus in this paper—with the main concern being loss or reduced expression of the transgenic insert [ 4 , 42 ]. If such a vaccine were to evolve fast enough or long enough that it lost the insert, vaccine efficacy might well suffer. We find that evolution during manufacture pre-host evolution can play a more important role than within-host evolution in reducing vaccine efficacy, and furthermore that it may be the more easily mitigated.
We developed and analyzed models to explore ways in which vaccine evolution could lead to a reduction in vaccine efficacy. An intrinsic fitness advantage of the revertant virus, expected because transgene expression is likely to have metabolic and other costs, will lead to vaccine being gradually overgrown by revertant. This is only likely to cause a reduction in the immunity to the vaccine antigen if it leads to a reduction in the absolute amount as opposed to merely a reduction in relative frequency of the vaccine virus.
There are in fact several mechanisms by which an ascending revertant population may suppress vaccine: revertant can reduce the amount of the vaccine virus in the host if the revertant uses resources required for virus replication or if the vaccine virus is cleared by the innate or adaptive responses elicited by the revertant. The clear and positive message from our study is that vaccine evolution, if it proves to be a problem for immunization, should be easily mitigated by manipulating the vaccine inoculum.
Critical to understanding and addressing this problem is recognizing that the vaccine may evolve both within the host and also during manufacture, whereby the inoculum already carries modest to high levels of revertant. The composition of the inoculum can have a large effect on within-host evolution and immunity. By limiting the amount of revertant in the inoculum, and also by boosting the inoculum level, it should usually be possible to limit the amount of within-host vaccine evolution and ensure that immunization is effective.
We emphasize, however, that this solution will typically not work for transmissible vaccines and vaccines that establish long term infections within the host. Furthermore, using a large inoculum may seem to defeat the purpose of using a live vaccine. There may be cases in which vaccine evolution is so rapid that controlling the inoculum is not sufficient. The solution in this case is to develop or engineer the vaccine with less of a disadvantage.
The timing and tissues of antigen expression, location of the transgene in the vector genome, and the size of the transgene may all influence intrinsic fitness effects [ 10 , 11 , 19 , 43 , 44 ]. Directed evolution approaches might also improve vaccine efficacy: one simple approach in reducing an intrinsic cost might be to adapt the vector in vitro to host cells expressing the antigen in trans , allowing compensatory mutations to evolve in response to the antigen before the transgene is cloned into the genome.
This adapted vector would then be used as the vaccine backbone. Another simple approach would be to compete several different vaccine designs in vitro and pick the design with highest retention of the transgene. Any approach using in vitro adaptation needs to avoid adapting the vector to the extent that it compromises ability to grow in vivo.
Most of these possibilities are ways to reduce pre-host evolution and reduce revertant concentration in the inoculum. One may hope that vaccine designs which reduce pre-host evolution also reduce within-host evolution. Measuring the intrinsic fitness effect of the transgene is likely to be an important step in vaccine design.
For assessing vaccine evolution, the relevant biological realms are within the host and in vitro. There are various ways intrinsic fitness effects and their evolutionary consequences might be studied.
Vaccine growth in tissue culture may reveal some aspects of intrinsic fitness effects and should be relatively easy to study. Deletion of the transgene per se would be detectable by PCR, and the fitness advantage of revertant over vaccine could be measured from changes in revertant frequency.
The quantitative relevance of an in vitro estimate to in vivo growth would be unknown, but the measure should allow qualitatively comparing engineering designs that improve intrinsic vaccine fitness.
If vaccine reversion were due to down regulation of the transgene instead of deletion, fitness estimation would require knowing the mutations responsible and monitoring their frequencies. Use of culture-wide antigen levels to measure fitness might provide a sense of whether vaccine evolution would lead to reduced antigen levels in vivo , but it would be less sensitive in measuring evolution than is measuring mutation frequencies.
Evolution is not the only consideration in designing a recombinant vector vaccine, and the model helps us identify vaccine properties that promote efficacy. First the vaccine should elicit an immune response that rapidly clears the pathogen i. Second, the vaccine should elicit a large response to this antigen.
This requires that the antigen rapidly elicits immunity i. Engineering this requires tackling a trade-off between avoiding vaccine clearance i. Vaccines designed to express the antigen in a form that is different from that in the pathogen might help solve this problem. Thus, to elicit immunity to influenza, one might design secreted forms of the hemagglutinin or neuraminidase proteins.
A recombinant hemagglutinin protein that is secreted rather than on the virion surface would prevent the antibody response to this protein from clearing the recombinant vector vaccine have low k X without compromising the clearance of the influenza virus pathogen which has hemagglutinin on its surface i.
In this manner our model allows the identification and tuning of parameters that affect vaccine efficacy, and a comprehensive search of parameter space would identify ideal combinations of vaccine properties. In vitro assays may be useful in measuring intrinsic fitness effects, but in vivo —in the patient—is the ultimate environment for studying within-host evolution and its effects.
Not only are the dynamics of viral spread different between in vitro and in vivo environments, but most immune components will be in play only in vivo. Furthermore, those components may vary across tissues within the host. Sampling across this heterogeneity in vivo will be challenging but may be necessary to know whether, when, and where vaccine evolution is a problem.
If revertant remains a minority of the population, we expect that vaccine evolution can be ignored. Perhaps in vitro studies of vaccine evolution will provide most of the information relevant to in vivo evolution, but it is too early to know. We have focused on recombinant vector vaccines that cause acute infections. Necessarily, our recommendations are based on simple models that are caricatures of the complex within-host dynamics of acute infections.
Simple models are appropriate at this stage because of uncertainties at many biological levels, and under these circumstances simple models frequently generate more robust results than do complex models [ 45 , 46 ]. The generation of innate and adaptive responses can be modeled with different assumptions than used here, and those alternative processes may affect the conclusions.
For example, time-lags in the activation of cells may dominate the time for the generation of an innate immune response, with virus density having a consequently smaller role than assumed here as can be seen in [ 47 ] and modeled in [ 30 ].
We have modeled that responses to different antigens are generated independently of each other and do not compete. We have done so because vaccines are likely to cause relatively mild infections during which the densities of pathogen and immune cells do not reach sufficiently high levels required for competitive interactions to be important.
The adaptive immune response may be more influenced by recruitment which is followed by a period of proliferation even in the absence of antigen [ 48 — 50 ]. Both these scenarios would minimize the impact of evolutionary changes in the vaccine on the amount of immunity generated to the transgene. Finally, it is easily appreciated that there are realms we do not consider, such as within-host spatial structure [ 51 ] and recombinant vector vaccines based on viruses such as cytomegalovirus that cause persistent infections [ 52 ] or that are transmissible.
Spatial structure may limit the impact of vaccine evolution on immunity e. In contrast, vaccines that cause persistent infections or are transmissible are likely to be more severely affected by evolution than are vaccines causing acute infections, as there is a longer timeframe for evolution to operate.
With so little experience from recombinant vector vaccines, we can merely guess how commonly the neglect of within-host evolution will compromise vaccine efficacy.
Given that simple steps can be taken to reduce vaccine evolution, vaccine development programs should at least entertain the possibility that evolution can underlie failure. Avoiding vaccine evolution may be easier than developing an entirely new vaccine. This file was used to generate the figures in the paper but may also be used to run other conditions, such as the case when resource limitation controls the infection.
The S1 File allows easy modification to explore other parameter values, so the figures generated here do not represent a thorough coverage of parameter space. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
National Center for Biotechnology Information , U. PLoS Comput Biol. Published online Jul James J. Scott L. Author information Article notes Copyright and License information Disclaimer. Received Feb 6; Accepted Jun 7. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
This article has been cited by other articles in PMC. S1 File: R Markdown file of code to run numerical trials of equations and generate figures. Rmd 30K. S2 File: Mathematica code to run numerical trials of equations. S1 Fig: Additional figures generated by the existing S1 File. Abstract Replicating recombinant vector vaccines consist of a fully competent viral vector backbone engineered to express an antigen from a foreign transgene.
Author summary Recombinant vector vaccines are live replicating viruses that are engineered to carry extra genes derived from a pathogen—and these extra genes produce proteins against which we want to generate immunity. Introduction Live vaccines replicate within the host.
Table 1 Consequences of evolution for traditional live attenuated and recombinant vector vaccines. Factor Attenuated vaccine Recombinant-vector vaccine type of evolution reversion toward wild-type loss of insert virulence higher virulence little change in virulence immunity possible increase possible reduction transmission increase no effect or increase. Open in a separate window. Methods Our models are numerical analyses of ordinary differential equations.
Two realms of vaccine evolution Vaccine evolution can be inimical to immunization by limiting vaccine antigen levels in the host. Fig 1. Impact of pre-host evolution on within-host evolution. A short duration of infection limits within-host evolution Any fitness advantage of revertant means that its frequency—its abundance relative to the vaccine—will increase during active viral growth Fig 2.
Fig 2. Independent growth of vaccine blue and revertant green. Evolution versus immunity Surprisingly, vaccine evolution per se need not reduce the immune response, even when its magnitude is large. Numbers versus frequencies Models of evolution often address relative frequencies, on a scale of 0 to 1. Bases and consequences of vaccine inferiority and interference Intrinsic fitness differences Intrinsic fitness effects are considered here to be those that stem from the intracellular processes of viral gene expression and assembly, independent of host immune responses.
Three mechanisms of vaccine-revertant interference Fig 2 presented a hypothetical case in which evolutionary superiority of revertant did not suppress vaccine growth, hence evolution had little effect on antigen production. Adaptive immunity to the vaccine antigen may also contribute to vaccine inferiority—and feed back to inhibit itself The preceding paragraphs omitted adaptive immunity to the antigen.
Beyond intuition: A formal model and numerical results We now employ quantitative models to evaluate the intuitive ideas presented above. Fig 3. Diagram of model processes and interactions. Evolution can matter In the trials used for illustration, we allow innate immunity to control the infection and adaptive immunity to cause final clearance. Fig 4. Representative dynamics contrasting vaccine evolution with no evolution. Fig 5. Viral load and the level of immunity to the vaccine antigen depend on evolution and vaccine composition pre-host evolution.
Vaccine evolution driven by adaptive immunity We focus on infections of short duration—that are cleared and do not rebound once suppressed. Fig 6. Effect of evolution on the suppression of immunity by impairment parameters.
Escaping the effects of evolution: Manipulate the inoculum The results above suggest that vaccine evolution is only likely to compromise immunity if there is substantial pre-host or within-host evolution and if this evolution depresses vaccine virus in the host. Fig 7. Effects of manipulating the inoculum on immunity to the vaccine. Discussion Any live viral vaccine may evolve within the host.
Supporting information S1 Appendix Equations and parameters. PDF Click here for additional data file. S1 File R Markdown file of code to run numerical trials of equations and generate figures.
RMD Click here for additional data file. S2 File Mathematica code to run numerical trials of equations. NB Click here for additional data file. S1 Fig Additional figures generated by the existing S1 File. Data Availability All relevant data are within the manuscript and its Supporting Information files or can be generated by the Supplement software.
References 1. Bull JJ. Evolutionary reversion of live viral vaccines: Can genetic engineering subdue it? Virus Evolution. Hanley KA. The double-edged sword: How evolution can make or break a live-attenuated virus vaccine. Vaccine-derived poliomyelitis 12 years after infection in Minnesota. The New England Journal of Medicine. Transmissible Viral Vaccines. Trends in Microbiology. Rationalizing the development of live attenuated virus vaccines. Nature biotechnology.
Stability of RNA virus attenuation approaches. Unique safety issues associated with virus-vectored vaccines: Potential for and theoretical consequences of recombination with wild type virus strains. Defining the interval for monitoring potential adverse events following immunization AEFIs after receipt of live viral vectored vaccines. Crow JF, Kimura M. It also avoids mutations that can occur when viruses are grown in eggs, which can sometimes affect how well the finished vaccine works.
A study in Hong Kong showed that recombinant flu vaccination resulted in a greater antibody response in adults 65 and older compared to traditional flu vaccines. Although increased antibody response following flu vaccination does not guarantee better clinical protection, additional studies to determine the possible benefits of recombinant flu vaccines in this age group are warranted. Recombinant flu vaccines production method does not require an egg-grown vaccine virus and does not use chicken eggs at all in the production process.
In clinical studies, the safety of recombinant flu vaccines was comparable to that of other injectable flu vaccines. The most common side effects reported after receipt of Flublok Quadrivalent were similar to those reported for other injectable vaccines and include pain and tenderness at the injection site, headache, fatigue, and muscle or joint aches. For more information see the Flublok Quadrivalent package insert external icon.
People who have a history of severe egg allergy those who have had any symptom other than hives after exposure to egg should be vaccinated in a medical setting, supervised by a health care provider who is able to recognize and manage severe allergic reactions. Two completely egg-free ovalbumin-free flu vaccine options are available: quadrivalent recombinant vaccine and quadrivalent cell-based vaccine.
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