Please read Imperfect Vaccination by Read et al i

RESEARCH ARTICLE

Imperfect Vaccination Can Enhance the Transmission of Highly Virulent Pathogens Andrew F. Read1,2*, Susan J. Baigent3, Claire Powers3, Lydia B. Kgosana3, Luke Blackwell3, Lorraine P. Smith3, David A. Kennedy1,2, Stephen W. Walkden-Brown4, Venugopal K. Nair3

1 Center for Infectious Disease Dynamics, Departments of Biology and Entomology, The Pennsylvania State University, University Park, Pennsylvania, United States of America, 2 Fogarty International Center, National Institutes of Health, Bethesda, Maryland, United States of America, 3 Avian Oncogenic Virus Group, The Pirbright Institute, Compton, Newbury, Berkshire, United Kingdom, 4 School of Environmental and Rural Science, The University of New England, Armidale, Australia

* [email protected]

Abstract Could some vaccines drive the evolution of more virulent pathogens? Conventional wisdom

is that natural selection will remove highly lethal pathogens if host death greatly reduces

transmission. Vaccines that keep hosts alive but still allow transmission could thus allow

very virulent strains to circulate in a population. Here we show experimentally that immuni-

zation of chickens against Marek's disease virus enhances the fitness of more virulent

strains, making it possible for hyperpathogenic strains to transmit. Immunity elicited by

direct vaccination or by maternal vaccination prolongs host survival but does not prevent

infection, viral replication or transmission, thus extending the infectious periods of strains

otherwise too lethal to persist. Our data show that anti-disease vaccines that do not prevent

transmission can create conditions that promote the emergence of pathogen strains that

cause more severe disease in unvaccinated hosts.

Author Summary

There is a theoretical expectation that some types of vaccines could prompt the evolution of more virulent (“hotter”) pathogens. This idea follows from the notion that natural selec- tion removes pathogen strains that are so “hot” that they kill their hosts and, therefore, themselves. Vaccines that let the hosts survive but do not prevent the spread of the patho- gen relax this selection, allowing the evolution of hotter pathogens to occur. This type of vaccine is often called a leaky vaccine. When vaccines prevent transmission, as is the case for nearly all vaccines used in humans, this type of evolution towards increased viru- lence is blocked. But when vaccines leak, allowing at least some pathogen transmission, they could create the ecological conditions that would allow hot strains to emerge and per- sist. This theory proved highly controversial when it was first proposed over a decade ago, but here we report experiments with Marek’s disease virus in poultry that show that modern commercial leaky vaccines can have precisely this effect: they allow the onward

PLOS Biology | DOI:10.1371/journal.pbio.1002198 July 27, 2015 1 / 18

OPEN ACCESS

Citation: Read AF, Baigent SJ, Powers C, Kgosana LB, Blackwell L, Smith LP, et al. (2015) Imperfect Vaccination Can Enhance the Transmission of Highly Virulent Pathogens. PLoS Biol 13(7): e1002198. doi:10.1371/journal.pbio.1002198

Academic Editor: Christophe Fraser, Imperial College London, UNITED KINGDOM

Received: January 15, 2015

Accepted: June 11, 2015

Published: July 27, 2015

Copyright: © 2015 Read et al. 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.

Data Availability Statement: All data files are deposited in Dryad, doi:10.5061/dryad.4tn48.

Funding: This work was funded by the Institute of General Medical Sciences, National Institutes of Health (R01GM105244) and by UK Biotechnology and Biological Sciences Research Council as part of the joint NSF-NIH-USDA Ecology and Evolution of Infectious Diseases program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

transmission of strains otherwise too lethal to persist. Thus, the use of leaky vaccines can facilitate the evolution of pathogen strains that put unvaccinated hosts at greater risk of severe disease. The future challenge is to identify whether there are other types of vaccines used in animals and humans that might also generate these evolutionary risks.

Introduction Infectious agents can rapidly evolve in response to health interventions [1]. Here, we ask whether pathogen adaptation to vaccinated hosts can result in the evolution of more virulent pathogens (defined here to mean those that cause more or faster mortality in unvaccinated hosts).

Vaccination could prompt the evolution of more virulent pathogens in the following way. It is usually assumed that the primary force preventing the evolutionary emergence of more viru- lent strains is that they kill their hosts and, therefore, truncate their own infectious periods. If so, keeping hosts alive with vaccines that reduce disease but do not prevent infection, replica- tion, and transmission (so-called “imperfect” vaccines) could allow more virulent strains to cir- culate. Natural selection will even favour their circulation if virulent strains have a higher transmission in the absence of host death or are better able to overcome host immunity. Thus, life-saving vaccines have the potential to increase mean disease virulence of a pathogen popula- tion (as assayed in unvaccinated hosts) [2–4].

The plausibility of this idea (hereafter called the “imperfect-vaccine hypothesis”) has been confirmed with mathematical models [2,5–9]. Efficacy and mode of action are key. If the vac- cine is sterilizing, so that transmission is stopped, no evolution can occur. But if it is non-steril- izing, so that naturally acquired pathogens can transmit from immunized individuals (what we hereafter call a “leaky” vaccine), virulent strains will be able to circulate in situations in which natural selection would have once removed them [2]. Thus, anti-disease vaccines (those reduc- ing in-host replication or pathogenicity) have the potential to generate evolution harmful to human and animal well-being; infection- or transmission-blocking vaccines do not [2–9]. Note that the possibility of vaccine-driven virulence evolution is conceptually distinct from vaccine- driven epitope evolution (antigenic escape), in which variants of target antigens evolve because they enable pathogens that are otherwise less fit to evade vaccine-induced immunity. The evo- lution of escape variants has been frequently observed [4,10].

The imperfect-vaccine hypothesis attracted controversy [11–14], not least because human vaccines have apparently not caused an increase in the virulence of their target pathogens. But most human vaccines are sterilizing (transmission-blocking) or not in widespread use or only recently introduced [4]. Moreover, unambiguous comparisons of strain virulence and the impact of vaccination on transmission require experimental infections in the natural host— clearly impossible for human diseases. The situation is different for veterinary infections. Here, we report experiments with Marek’s disease virus (MDV), a highly contagious oncogenic her- pesvirus that costs the global poultry industry more than $US2 billion annually [15]. We test a key prediction of the imperfect-vaccine hypothesis: that vaccination will elevate the fitness of highly virulent strains above that of less virulent strains.

Chickens become infected with MDV by inhalation of dust contaminated with virus shed from the feather follicles of infected birds. In a contaminated poultry house, chicks are infected soon after hatching and remain infectious for life [16]. The virus can remain infectious in poul- try dust for many months [17,18]. As originally described, Marek’s disease (MD) was a paraly- sis of older birds, but by the 1950s, “acute MD” characterised by lymphomas in multiple

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Abbreviations: c.i., confidence interval; MD, Marek’s disease; MDV, Marek’s disease virus; RIR, Rhode Island Red; SPF, Specific pathogen-free; VCN, virus genome copy number.

organs in younger birds was occurring. This became the dominant form of MD, with increas- ing virulence, characterised by more severe lymphomas and mortality at increasingly early ages and, under some circumstances, paralysis and death in the first weeks of life, well before lym- phoma formation [15,19].

MDV has been evolving in poultry immunized with leaky anti-disease vaccines since the introduction of the first vaccines in 1970 [15,19–24]. All MD vaccines are live viruses adminis- tered to 18-day-old embryos or immediately after hatch, and vaccinated birds can become infected and shed wild-type virus [25–28]. Wild-type MD viruses are so-called serotype 1 viruses. First-generation vaccines include a serotype 3 herpesvirus of turkeys called HVT; sec- ond-generation vaccines are a combination of HVT and SB-1, a serotype 2 isolate. Third-gen- eration vaccines are based on an attenuated serotype-1 virus isolate CVI998, the so-called Rispens vaccine [15,19–24].

Results

Viral Shedding Our first three experiments involved Rhode Island Red (RIR) chickens, a breed that has not been subject to the intensive selective breeding and outcrossing that characterizes modern commercial chicken strains. Specific pathogen-free (SPF) parent birds were unvaccinated, and so offspring used in our first two experiments were free from maternally derived antibodies. In our first experiment, we infected 8-d-old chicks with five strains of MDV chosen to span the virulence spectrum defined by Witter and colleagues [21,29]. The viral strains varied from the less virulent HPRS-B14, which killed 60% of unvaccinated birds over 2 mo, to the highly lethal Md5 and 675A, which killed all unvaccinated birds in 10 d (Fig 1, top panels). When age- matched birds were vaccinated 8 d earlier with HVT, the first MDV vaccine to go into com- mercial use, survival improved dramatically, with a few deaths occurring only late in the exper- iment, and then only in birds infected with the most virulent strains (Fig 1, top panels).

We collected dust from the isolators containing infected birds and measured the concentra- tion of virus genomes in the dust using real-time PCR. At contemporaneous time points, vacci- nated birds shed fewer virus genome copies than unvaccinated birds infected with the same viral strain (Fig 1, middle panels). Those patterns reflected viral loads in the feather follicles (S1 Fig). Critically, the infectious period of unvaccinated birds infected with our two most virulent strains was less than a week because hosts died so rapidly. During that week, barely any virus was shed (Fig 1, middle panels). In contrast, the infectious period of the least virulent strains continued for the entire experiment (almost 2 mo). Thus, the least virulent strain shed several orders of magnitude more virus from unvaccinated birds than did the virulent strains (Fig 1, bottom panels). By preventing death, vaccination greatly increased the infectious period of the most virulent strains, increasing the total amount of virus shed by several orders of magnitude, and increasing it above that of the least virulent strain (Fig 1, bottom panels). Thus, the net effect of vaccination on both host survival rates and daily shedding rates was to vastly increase the amount of virus shed by virulent strains into the environment.

Onward Transmission To confirm that virus shed into the environment was a robust proxy for overall bird-to-bird transmission potential, we co-housed birds infected with our three most virulent strains with immunologically-naïve sentinel birds (Experiment 2). When unvaccinated birds were infected with the two most lethal strains (Md5 and 675A), they were all dead within 10 days (Fig 2A), before substantial viral shedding had begun (S2 Fig). Consequently, no sentinel birds in those isolators became infected (Fig 2B) and none died (Fig 2C). In contrast, when HVT-vaccinated

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birds were infected with either of those hyperpathogenic strains, they survived for 30 days or more (Fig 2A), allowing substantial viral shedding (S2 Fig). All co-housed sentinels conse- quently became infected (Fig 2B) and went on to die as a result of MDV infection (Fig 2C). Thus, in accordance with the imperfect-vaccine hypothesis, vaccination enabled the onward transmission of viruses otherwise too lethal to transmit, putting unvaccinated individuals at great risk of severe disease and death.

Interestingly, the viral strain 595 was slightly less virulent than the other two viruses (taking a day longer to kill half of the unvaccinated birds, and 6 d longer to kill them all) (Fig 2A). This slightly reduced mortality rate prolonged the viral shedding from unvaccinated birds, so that about 100-fold more virus was shed into the environment by the 595-infected cohort than from the cohorts infected by the two more lethal strains (S2 Fig). This was evidently sufficient for transmission, because all co-housed sentinels eventually became infected (Fig 2B) and went on to die (Fig 2C). Thus, slight reductions in lethality can be sufficient to allow onward transmission. Nonetheless, even for strain 595, vaccination led to more rapid infection of senti- nels (Fig 2B; median time to positivity 9 d earlier than in unvaccinated birds, p < 0.05), thus increasing the rate at which secondary cases were generated, a critical determinant of both viral fitness and case incidence in a rising epidemic.

Fig 1. Impact of vaccination on mortality and viral shedding of five strains of MDV. Experiment 1. Groups of 20 Rhode Island Red chickens were unvaccinated (dotted lines, light shading) or HVT-vaccinated (solid lines, dark shading) at 1 d of age and challenged with viral strains HPRS-B14 (black), 571 (purple), 595 (green), Md5 (blue), or 675A (red) 8 d later. Viral strains vary in virulence in unvaccinated hosts, and vaccination protects against death (top panels, with strains arranged in order of increasing virulence from left to right.). Vaccination suppresses the concentration of virus in dust, but by keeping hosts alive, prolongs the infectious periods of hyperpathogenic MDV (middle panels). This means that cumulative number of virus genome copies (VCN) shed per bird is suppressed by vaccination for the least virulent strain and enhanced by several orders of magnitude for the most virulent (bottom panels). Error bars and shaded regions indicate 95% confidence interval (c.i.) Raw data can be found at http://dx.doi.org/10.5061/dryad.4tn48.

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Maternally Derived Antibody The high mortality rates we observed in unvaccinated chickens infected with our most virulent strains are due to early mortality syndrome, which involves the rapid onset of paralysis, dis- orientation and an inability to feed and move, followed by death [30–33]. In today’s modern industry, parental birds are almost always vaccinated against MDV, which results in the trans- fer of maternal antibody to chicks. These antibodies appear to be protective against the early mortality syndrome [30–33]. This raises the prospect that vaccination of laying hens could also permit onward transmission of viral strains that would be too lethal to otherwise transmit from offspring birds. We tested this possibility with further experiments using our most (675A) and least (HPRS-B14) virulent virus strains, again in Rhode Island Red birds, but this time

Fig 2. Vaccination enhances transmission of hyperpathogenic MDV. Experiment 2. Groups of ten birds were HVT-vaccinated (solid lines) or not (dotted lines) and experimentally infected with one of our three most virulent MDV strains, 595 (green), Md5 (blue) and 675A (red), and co-housed with ten unvaccinated sentinel birds. Vaccination prolonged the survival of experimentally infected birds (A), ensuring that sentinel birds became infected (B) and, hence, died (C). In B and C, solid lines denote sentinels cohoused with vaccinated experimentally infected birds and dotted lines denote sentinels cohoused with unvaccinated experimentally infected birds. Raw data can be found at http://dx.doi.org/10.5061/dryad.4tn48.

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including chicks derived from hens vaccinated 4–5 wk prior to egg lay with a standard com- mercial Rispens vaccine (Experiment 3).

Vaccination of hens enhanced the survival of offspring experimentally infected with HRPS-B14 (Fig 3A, p < 0.05). Maternally derived antibody had no detectable effect on the rep- lication of that viral strain in the feather tips (S3 Fig panel A, p > 0.05) and, while it somewhat suppressed the amount of infectious virus shed into the environment early in infections (Figs 3B and S3B), it did not affect the rate at which sentinel birds became infected with HRPS-B14 (Fig 3C, p > 0.05) and few sentinels died (Fig 3D). Thus maternal protection had little impact on the transmission success of our least virulent strain.

However, presence of maternal antibody greatly impacted the transmission success of the most virulent strain (675A). As expected, the offspring of vaccinated hens survived for lon- ger following infection with 675A virus than did maternally derived antibody-negative chicks (Fig 3A, p < 0.05). As we found in our first two experiments, very little of the highly virulent strain was shed from birds with no immune protection before they died (Figs 3B and S3). Consequently, no sentinels became infected (Fig 3C). But birds with maternal protection sur- vived longer to shed more virus (Figs 3A, 3B, and S3B), so that all sentinel birds became infected (Fig 3C) and died (Fig 3D). Maternal vaccination was not as protective as direct vacci- nation of offspring (cf. Fig 3A with Fig 2A and the top panels of Fig 1). Nonetheless, vaccina- tion of laying hens, like the vaccination of offspring, enabled the onward transmission of the

Fig 3. Maternal vaccination enhances viral shedding and onward transmission of hyperpathogenic MDV. Experiment 3. Groups of ten unvaccinated chicks produced by hens that were Rispens-vaccinated (solid lines) or not (dotted lines) were infected with viral strains HPRS-B14 (black) or 675A (red) and cohoused with sentinels of the same maternal antibody status. Maternally derived antibodies prolonged the survival of experimentally infected birds (A) and enhanced the amount of virus shed into the environment by the hyperpathogenic strain (B), making possible the infection of sentinels with the most virulent strain (C), which led to their death (D). Shaded regions represent 95% c.i. for unvaccinated (light) and vaccinated (dark). Raw data can be found at http://dx. doi.org/10.5061/dryad.4tn48.

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hyperpathogenic strain from offspring (Fig 3C). Again, these data are consistent with the imperfect-vaccine hypothesis.

Transmission between Commercial Birds Our experiments above show that direct vaccination of birds or vaccination of parent hens makes possible the onward transmission of viral strains otherwise too lethal to transmit, and thus that unvaccinated individuals are put at increased risk of severe disease and death. How- ever, in a modern commercial broiler setting, all birds in a flock would originate from vacci- nated hens (and so would be positive for maternally derived antibody), and also be vaccinated. We thus set out to determine whether our most virulent strain could transmit to vaccinated sentinels, a necessary condition for persistence of hyperpathogenic strains in the modern industry (Experiment 4). To mimic the current commercial broiler situation, we obtained modern commercial broiler birds derived from Rispens-vaccinated hens and, at 1 d of age, we HVT-vaccinated all the birds we would experimentally infect. Those birds were then infected with our most virulent viral strain (675A) at 8 d of age. We cohoused those experimentally infected birds with sentinel birds, which were either HVT vaccinated or not. We performed this experiment twice. To accommodate changing regulatory requirements (see Methods), we did the first replicate with birds housed in isolators until 35 d of age, after which they were moved to floor until they were 11 wk old (Experiment 4a), and the second replicate with birds maintained in floor pens from 1 d of age until 7 wk of age (Experiment 4b).

All sentinels became infected, irrespective of vaccine status (Fig 4A). Thus, vaccinated maternal antibody positive commercial birds shed wild-type virus that caused infections in both vaccinated and unvaccinated maternal antibody positive birds. Vaccination only slightly suppressed viral replication in the infections acquired by the sentinel birds (Fig 4B). Impor- tantly, all sentinels, vaccinated and unvaccinated, became virus positive in the feather follicles, meaning that they themselves started shedding. Vaccination protected sentinel birds from death (Fig 4C), prolonging infectious periods by about 2 wk (Fig 4D; standard error of the dif- ference ±3.2 d, F1,36 = 19.9, p < 0.0001). Thus, not only does our most virulent strain transmit between modern commercial broilers when they are vaccinated, the duration of shedding in the next step in the transmission chain is also increased by vaccination.

Discussion MDV became increasingly virulent over the second half of the 20th century [19,21–24]. Until the 1950s, strains of MDV circulating on poultry farms caused a mildly paralytic disease, with lesions largely restricted to peripheral nervous tissue. Death was relatively rare. Today, hyper- pathogenic strains are present worldwide. These strains induce lymphomas in a wide range of organs and mortality rates of up to 100% in unvaccinated birds. So far as we are aware, no one has been able to isolate non-lethal MDV strains from today’s commercial (vaccinated) poultry operations [19,23]. Quite what promoted this viral evolution is unclear. The observation that successively more efficacious vaccines have been overcome by successively more virulent viral strains has prompted many MDV specialists to suggest that vaccination might be a key driver [19–24,34–37], though identifying the evolutionary pressures involved has proved challenging. There is no evidence in Marek’s disease that vaccine breakthrough by more virulent strains has anything to do with overcoming strain-specific immunity (e.g., epitope evolution); genetic and immunological comparisons of strains varying in virulence suggest that candidate virulence determinants are associated with host–cell interactions and viral replication, not antigens [19]. The imperfect-vaccine hypothesis was suggested as an evolutionary mechanism by which immunization might drive MDV virulence evolution [2], but there has been no experimental

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confirmation. Our data provide that: by enhancing host survival but not preventing viral shed- ding, MDV vaccination of hens or offspring greatly prolongs the infectious periods of hyper- pathogenic strains, and hence the amount of virus they shed into the environment.

Our data do not demonstrate that vaccination was responsible for the evolution of hyper- pathogenic strains of MDV, and we may never know for sure why they evolved in the first place. Clearly, many potentially relevant ecological pressures on virulence have changed with the intensification of the poultry industry. For instance, as the industry has expanded, broilers have become a much larger part of the industry, and broiler lifespans have halved with advances in animal genetics and husbandry; all else being equal, this would favour more viru- lent strains [28], so too might greater genetic homogeneity in flocks [38] or high-density rear- ing conditions [13], or indeed increased frequencies of maternally derived antibody if natural MDV infections became more common as the industry intensified in the pre-vaccine era (Fig 3) [39]. But whatever was responsible for the evolution of more virulent strains in the first place (and there may be many causes), our data show that vaccination is sufficient to maintain hyperpathogenic strains in poultry flocks today. By keeping infected birds alive, vaccination substantially enhances the transmission success and hence spread of virus strains too lethal to

Fig 4. Transmission of hypervirulent MDV to modern commercial birds. Experiment 4. Groups of ten modern commercial broiler chicks derived from Rispens-vaccinated hens were HVT-vaccinated at 1 d of age and experimentally infected with the hypervirulent MDV strain 675A. Those experimentally infected birds were co-housed with groups of ten sentinel birds from the same commercial stock (and thus also derived from Rispens-vaccinated hens) which were HVT-vaccinated (solid lines) or not (dotted lines). The experiment was performed twice (experiment 4a, red; experiment 4b, blue). Independent of their vaccine status, all sentinel birds became infected (A), with high levels of virus replication in feather follicles (B). Vaccination prolonged the survival of sentinel birds (C), and consequently their infectious period (D). In panel D, “x” denotes death because of MDV. Error bars, 95% c.i. Raw data can be found at http://dx. doi.org/10.5061/dryad.4tn48.

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persist in unvaccinated populations, which would therefore have been removed by natural selection in the pre-vaccine era.

The relaxation of natural selection against hyperpathogenic strains revealed by our experi- ments arises because vaccination enhances host survival. In serial passage experiments with a rodent malaria, immunity induced by whole parasite immunization [40] or vaccination with a recombinant antigen [10] also promoted the evolution of virulence. However, by design, those experiments did not allow host death to impact pathogen fitness, and so the evolution towards increased virulence was driven in a different way. Evidently, immunity in that system is dispro- portionately efficacious against less virulent strains. Our MDV experiments were not designed to test for within-host selection, but there is some sug

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