TWiV 348: Chicken shift

On episode #348 of the science show This Week in Virology, Vincent and Rich discuss fruit fly viruses, one year without polio in Nigeria, and a permissive Marek’s disease viral vaccine that allows transmission of virulent viruses.

You can find TWiV #348 at www.microbe.tv/twiv.

Permissive vaccines and viral virulence

chicken farmA permissive vaccine prevents disease in the immunized host, but does not block virus infection. Would a permissive vaccine lead to the emergence of more virulent viruses?

This hypothesis is based on the notion that viruses which kill their hosts too quickly are not efficiently transmitted, and are therefore removed by selection. However a vaccine that prevents disease, but not viral replication in the host, would allow virulent viruses to be maintained in the host population. It has been suggested that in this scenario, viruses with increased virulence would be selected if such a property aids transmission between hosts.

On the surface this hypothesis seems reasonable, but in my opinion it is flawed. One problem is that increased transmission might not always be associated with increased virulence. The more serious flaw lies in making anthropomorphic assessments of what we think viruses require, such as concluding that increased viral transmission is a desired trait. Our assumptions fail to recognize the main goal of evolution: survival. Evolution does not move a virus along a trajectory aimed at perfection. Change comes about by eliminating those viruses that are not well adapted for the current conditions, not by building a virus that will fare better tomorrow. All the viruses on Earth today transmit well enough, or they would not be here; yet some kill their hosts clearly much faster than others. The fact is that humans have little understanding of what drives virus evolution in large populations. Our assumptions of what constitute the selective forces are usually tainted by anthropomorphism.

This long preamble is an introduction to a series of findings which are purported to support the idea that permissive vaccines (the authors call them ‘leaky’ and ‘imperfect’ vaccines but I dislike both names because they imply defects) can lead to the selection of more virulent viruses. The subject of the paper is Marek’s disease virus (MDV), a herpesvirus that infects chickens. MDV is shed from feather follicles of infected chickens and is spread to other birds when then inhale contaminated dust. Vaccines have been used to prevent MDV infection since the early 1970s. These vaccines prevent disease, but do not block viral replication, and vaccinated, infected birds can shed wild type virus. The virulence of MDV has been increasing since the 1950s, initially from a paralytic disease, to paralysis and death. The authors wonder if the use of permissive Marek’s vaccines has lead to the selection of more virulent viruses.

To address their hypothesis, the authors inoculate vaccinated or unvaccinated chickens with a series of MDV isolates that range from low to high virulence. Unvaccinated chickens inoculated with the most virulent MDV died within a week and shed little virus. In contrast, most vaccinated birds survived infection with virulent viruses, and shed virus for the length of the experiment, 56 days.

A transmission experiment was done to determine if shed virus could infect other birds. The authors infected vaccinated or unvaccinated birds and asked if sentinel, unvaccinated chickens became infected. Unvaccinated birds died within 10 days after infection with virulent MDV, and did not transmit infection. In contrast, vaccinated birds survived at least 30 days, and co-housed sentinel animals became infected and died.

The experiments are well done and the conclusions are clear: more virulent Marek’s disease viruses replicate longer in vaccinated than unvaccinated chickens, and can be readily transmitted to other chickens. But these results do not prove that more virulent MDV arose because of permissive vaccines. Nor do the results prove in general that leaky vaccines lead to selection of more virulent viruses. The results simply show that a vaccine that does not prevent replication will allow transmission of virulent viruses.

To prove that vaccinated chickens can allow the selection of more virulent viruses, vaccinated chickens could be infected with an avirulent virus, and the shed virus collected and used to infect additional, vaccinated birds. This process could be repeated to determine if more virulent viruses arise. While the results of this gain-of-function experiment would be informative, they would be done in a controlled laboratory setting which would not duplicate all the selective forces present on a poultry farm.

The authors note that most human vaccines do prevent replication of infecting virus. They do not mention the one important exception: the Salk poliovirus vaccines. People who are immunized with the Salk vaccine can be infected with poliovirus, which will then replicate in the intestines, be shed in the feces, and transmitted to others. This behavior has been well documented in human populations, yet the virulence of poliovirus has not increased for the 60 years during which the Salk vaccine has been used.

I do not feel that these experimental results have general implications for the use of any animal vaccine. It is unfortunate that the work has been covered in many news sources with the incorrect implication that vaccines may be responsible for the emergence of more virulent viruses.

Transgenic pigs resistant to foot-and-mouth disease

Foot-and-mouth disease virus (FMDV) infects cloven-hoofed animals such as cattle, pigs, sheep, goats, and many wild species. The disease caused by this virus is a substantial problem for farmers because infected animals cannot be sold. Transgenic pigs have now been produced which express a short interfering RNA (siRNA) and consequently have reduced susceptibility to infection with FMDV.

FMDV is classified in the picornavirus family which also contains poliovirus and rhinoviruses. The virus is highly contagious and readily spreads long distances via wind currents, and among animals by aerosols and contact with farm equipment. Infection causes a high fever and blisters in the mouth and on the feet – hence the name of the disease. When outbreaks occur, they are economically devastating. The 2001 FMDV outbreak in the United Kingdom was stopped by mass slaughter of all animals surrounding the affected areas – an estimated 6,131,440 – in less than a year.

Vaccines against the virus can be protective but they are not an optimal solution. One problem is that antigenic variation of the virus may thwart protection. In addition, countries free of FMDV generally do not vaccinate because this practice would make the animals seropositive and prevent their export (it is not possible to differentiate between antibodies produced by natural infection versus immunization). Furthermore, if there were an outbreak of foot-and-mouth disease in such countries, the rapid replication and spread of the virus would make vaccination ineffective – hence culling of animals as described above is required. Clearly other means of protecting animals against FMDV are needed.

Synthetic short interfering RNAs (siRNA) have been shown to block viral replication in cell culture and in animals. To achieve such inhibition, short synthetic RNAs complementary to viral sequences are produced in cells. Upon infection, these siRNAs combine with the cellular RNA-induced silencing complex (RISC) which then targets the viral RNA for degradation.

To determine if siRNA could be used to protect pigs from foot-and-mouth disease, a complementary viral sequence was first identified that blocks FMDV replication in cell culture by ~97%. A vector containing this siRNA sequence was then used to produce transgenic pigs. Such animals not only express the antiviral siRNA, but as the encoding vector is present in germ cells, it is passed on to progeny pigs.

Expression of the siRNA was confirmed in a variety of transgenic pig tissues, including heart, lung, spleen, liver, kidney, and muscle. In fibroblasts produced from transgenic pigs, virus replication was reduced 30 fold. When transgenic pigs were inoculated intramuscularly with FMDV, none of the animals developed signs of disease such as fever or blisters of the feet and nose. In contrast, control non-transgenic pigs developed high fever and lesions. Viral RNA levels in the blood of transgenic pigs were 100-fold lower than in control animals. At 10 days post-infection no viral RNA was detected in heart, lung, spleen, liver, kidney, and muscle, while high levels were observed in these organs from non-transgenic controls.

These results show that siRNAs can protect transgenic pigs from FMDV induced disease. An important question that must be answered is whether transgenic pigs still contain enough virus to transmit infection to other animals. In addition, siRNAs are short – 21 nucleotides – and a mutation in the viral genome can block their inhibitory activity. Therefore it would be important to determine if mutations arise in the FMDV genome that lead to resistance to siRNAs.

Even if transgenic siRNA pigs do not transmit infection, and viral resistance does not arise, I am not sure that consumers are ready to accept such genetically modified animals.

TWiV 341: Ebolavirus experiences

On episode #341 of the science show This Week in Virology, Vincent returns to the University of Glasgow MRC-Center for Virus Research and speaks with Emma, Gillian, and Adam about their ebolavirus experiences: caring for an infected patient, working in an Ebola treatment center in Sierra Leone, and making epidemiological predictions about the outbreak in west Africa.

You can find TWiV #341 at www.microbe.tv/twiv.

TWiV 335: Ebola lite

On episode #335 of the science show This Week in Virology, the TWiVumvirate discusses a whole Ebolavirus vaccine that protects primates, the finding that Ebolavirus is not undergoing rapid evolution, and a proposal to increase the pool of life science researchers by cutting money and time from grants.

You can find TWiV #335 at www.microbe.tv/twiv.

Blocking HIV infection with two soluble receptors

eCD4-FcBecause viruses must bind to cell surface molecules to initiate replication, the use of soluble receptors to block virus infection has long been an attractive therapeutic option. Soluble receptors have been developed that block infection with rhinoviruses and HIV-1, but these have not been licensed due to their suboptimal potency. A newly designed soluble receptor for HIV-1 overcomes this problem and provides broad and effective protection against infection of cells and of nonhuman primates.

Infection with HIV-1 requires two cell surface molecules, CD4 and a chemokine receptor (either CCR5 or CXCR4), which are engaged by the viral glycoprotein gp120 (illustrated). A soluble form of CD4 fused to an antibody molecule can block infection of most viral isolates, and has been shown to be safe in humans, but its affinity for gp120 is low. Similarly, peptide mimics of the CCR5 co-receptor have been shown to block infection, but their affinity for gp120 is also low.

Combining the two gp120-binding molecules solved the problem of low affinity, and in addition provided protection against a wide range of virus isolates. The entry inhibitor, called eCD4-Ig, is a fusion of the first two domains of CD4 to the Fc domain of an antibody molecule, with the CCR5-mimicking peptide at the carboxy-terminus (illustrated). It binds strongly to gp120, and blocks infection with many different isolates of HIV-1, HIV-2, SIV, and HIV-1 resistant to broadly neutralizing monoclonal antibodies. The molecule blocks viral infection at concentrations that might be achieved in humans (1.5 – 5.2 micrograms per milliliter).

When administered to mice, eCD4-Ig protected the animals from HIV-1. Rhesus macaques inoculated with an adenovirus-associated virus (AAV) recombinant containing the gene for eCD4-Ig were protected from infection with large amounts of virus for up to 34 weeks after immunization. Levels of eCD4-Ig in the sera of these animals ranged from 17 – 77 micrograms per milliliter.

These results show that eCD4-Ig blocks HIV infection with a wide range of isolates more effectively than previously studied broadly neutralizing antibodies. Emergence of HIV variants resistant to neutralization with eCD4-Ig would likely produce viruses that infect cells less efficiently, reducing their transmission. eCD4-Ig is therefore an attractive candidate for therapy of HIV-1 infections. Whether sustained production of the protein in humans will cause disease remains to be determined. Because expression of the AAV genome persists for long periods, it might be advantageous to include a kill-switch in the vector: a way of turning it off if something should go wrong.

TWiV 319: Breaking breakbone

On episode #319 of the science show This Week in Virology, the TWiVers review the outcomes of two recent phase 3 clinical trials of a quadrivalent dengue virus vaccine in Asia and Latin America.

You can find TWiV #319 at www.microbe.tv/twiv.

An unexpected benefit of inactivated poliovirus vaccine

Poliovirus by Jason Roberts

Poliovirus by Jason Roberts

The polio eradication and endgame strategic plan announced by the World Health Organization in 2014 includes at least one dose of inactivated poliovirus vaccine (IPV). Since 1988, when WHO announced the polio eradication plan, it had relied exclusively on the use of oral poliovirus vaccine (OPV). The rationale for including a dose of IPV was to avoid outbreaks of vaccine-derived type 2 poliovirus. This serotype had been eradicated in 1999 and had consequently been removed from OPV. However IPV, which is injected intramuscularly and induces highly protective humoral immunity, is less effective in producing intestinal immunity than OPV. This property was underscored by the finding that wild poliovirus circulated in Israel during 2013, a country which had high coverage with IPV. Furthermore, in countries that use only IPV, over 90% of immunized children shed poliovirus after oral challenge. I have always viewed this shortcoming of IPV as problematic, in view of the recommendation of the World Health Organization to gradually shift from OPV to IPV. Even if the shift to IPV occurs after eradication of wild type polioviruses, vaccine-derived polioviruses will continue to circulate because they cannot be eradicated by IPV. My concerns are now mitigated by new results from a study in India which indicate that IPV can boost intestinal immunity in individuals who have already received OPV.

To assess the ability of IPV to boost mucosal immunity, 954 children in three age groups (6-11 months, 5 and 10 years) were immunized with IPV, bivalent OPV (bOPV, containing types 1 and 3 only), or no vaccine. Four weeks later all children were challenged with bOPV, and virus shedding in the feces was determined 0, 3, 7, and 14 days later. The results show that 8.8, 9.1, and 13.5% of children in the 6-11 month, 5-year and 10-year old groups shed type 1 poliovirus in feces, compared with 14.4, 24.1, and 52.4% in the control group. Immunization with IPV reduced fecal shedding of poliovirus types 1 (39-74%) and 3 (53-76%). The reduction of shedding was greater after immunization with IPV compared with bOPV.

This study shows that a dose of IPV is more effective than OPV at boosting intestinal immunity in children who have previously been immunized with OPV. Both IPV and OPV should be used together in the polio eradication program. WHO therefore recommends the following vaccine regimens:

  • In all countries using OPV only, at least 1 dose of type 2 IPV should be added to the schedule.
  • In polio-endemic countries and in countries with a high risk for wild poliovirus importation and spread: one OPV birth dose, followed by 3 OPV and at least 1 IPV doses.
  • In countries with high immunization coverage (90-95%) and low wild poliovirus importation risk: an IPV-OPV sequential schedule when VAPP is a concern, comprising 1-2 doses of IPV followed by 2 or mores doses of OPV.
  • In countries with both sustained high immunization coverage and low risk of wild poliovirus importation and transmission: an IPV only schedule.

Type 2 OPV will be gradually removed from the global immunization schedules. There have been no reported cases of type 3 poliovirus since November 2012. If this wild type virus is declared eradicated later this year, presumably WHO will recommend withdrawal of type 3 OPV and replacement with type 3 IPV.

All 342 confirmed cases of poliomyelitis in 2014 were caused by type 1 poliovirus in 9 countries, mainly Pakistan and Afghanistan. Given the social and political barriers to immunization, it will likely take many years to eradicate this serotype.

TWiV 318: Last year in virology

On episode #318 of the science show This Week in Virology, the TWiV gang reviews ten fascinating, compelling, and riveting virology stories from 2014.

You can find TWiV #318 at www.microbe.tv/twiv.

Could Reston virus be a vaccine for Ebola virus?

crab eating macaqueI have received many questions about whether immunizing with Reston virus could protect against infection with Ebola virus. Usually the question comes together with the statement ‘because Reston virus does not cause disease in humans’. I can think of two reasons why a Reston virus vaccine is not a good idea.

There have been very few confirmed human infections with Reston virus (4 according to Fields Virology 6th Edition), and although these individuals did not show signs of disease, the number is too small to make any conclusions. For example, if the case fatality ratio of Reston virus in humans were 1%, we might not have yet seen any deaths due to the small number of confirmed infections. However if we were to immunize a million people with a Reston virus vaccine, and the case fatality ratio were 1%, there would be 10,000 deaths, obviously an unacceptable rate for a vaccine. As the virus causes disease in nonhuman primates, and there are so few human infections, it is not possible to know the case fatality ratio.

The other problem is that in general, infection with one of the Ebolaviruses does not confer protection against the others*. For example, animals that survive challenge with Ebola virus (Zaire) are not protected from infection with Sudan virus. For this reason, vaccines need to be prepared against representatives of all the species. I am not aware of any animal studies that have assessed whether Reston virus infection protects from infection with the other Ebolaviruses, but there is no reason to believe that such cross-protection would be achieved.

Vaccines are currently being tested against Ebola virus (species Zaire ebolavirus), cause of the outbreak in west Africa.

*Thanks to Andrea Marzi for information on cross-protection.