TWiV 371: Sympathy for the devil

TWiVOn episode #371 of the science show This Week in Virology, the TWiVologists discuss the finding of a second transmissible cancer in Tasmanian devils, and development of new poliovirus strains for the production of inactivated vaccine in the post-eradication era.

You can find TWiV #371 at www.microbe.tv/twiv.

Why do we still use Sabin poliovirus vaccine?

VAPPThe Sabin infectious, attenuated poliovirus vaccines are known to cause vaccine-associated paralysis in a small number of recipients. In contrast, the Salk inactivated vaccine does not cause poliomyelitis. Why are the Sabin vaccines still used globally? The answer to this question requires a brief visit to the history of poliovirus vaccines.

The inactivated poliovirus vaccine (IPV) developed by Jonas Salk was licensed for use in 1955. This vaccine consists of the three serotypes of poliovirus whose infectivity, but not immunogenicity, is destroyed by treatment with formalin. When prepared properly, IPV does not cause poliomyelitis (early batches of IPV were not sufficiently inactivated, leading to vaccine-associated outbreaks of polio, the so-called Cutter incident). From 1955 to 1960 cases of paralytic poliomyelitis in the United States dropped from 20,000 per year to 2,500.

While Salk’s vaccine was under development, several investigators pursued the production of infectious, attenuated vaccines as an alternative. This approach was shown to be effective by Max Theiler, who in 1937 had made an attenuated vaccine against yellow fever virus by passage of the virulent virus in laboratory mice. After many passages, the virus no longer caused disease in humans, but replicated sufficiently to induce protective immunity. Albert Sabin capitalized on these observations and developed attenuated versions of the three serotypes of poliovirus by passage of virulent viruses in different animals and cells. In contrast to Theiler’s yellow fever vaccine, which was injected, Sabin’s poliovirus vaccines were designed to be taken orally – hence the name oral poliovirus vaccine (OPV). As in a natural poliovirus infection, Sabin’s vaccines would replicate in the intestinal tract and induce protective immunity there and in the bloodstream.

Sabin began testing his attenuated vaccines in humans in 1954. By 1957 there was evidence that the virus that was fed to volunteers was not the same as the virus excreted in the feces. As Sabin writes:

It was evident, however, that as in the young adult volunteers, the virus in some of the stool specimens had a greater neurovirulence than the virus originally swallowed in tests in monkeys.

What Sabin did not know was whether the change in neurovirulence of his vaccine strains constituted a threat to the vaccine recipients and their contacts, a question that could only be answered by carrying out larger clinical trials. Many felt that such studies were not warranted, especially considering the success of IPV in reducing the number of paralytic cases. Sabin notes that his friend Tom Rivers, often called the father of American virology, told him to ‘discard the large lots of OPV that I had prepared into a suitable sewer’.

Despite the opposition to further testing of OPV in the US, others had different views. An international committee of the World Health Organization recommended in 1957 that larger trials of OPV should be carried out in different countries. Sabin’s type 2 vaccine was given to 200,000 children during an outbreak of polio in Singapore in 1958, and follow-up studies revealed no safety problems. In Czechoslovakia 140,000 children were given OPV and subsequent studies revealed that the virus spread to unimminized contacts but did not cause disease.

Perhaps the most important numbers came from trials of OPV in the Soviet Union. Sabin had been born in Russia and had close contacts with Soviet virologists, including Mikhail Chumakov, director of the Poliomyelitis Research Institute in Moscow. Chumakov was not satisfied with the results of IPV trials in his country and asked Sabin to send him OPV for testing. By the end of 1959 nearly 15,000,000 people had been given OPV in different parts of the Soviet Union with no apparent side effects. Dorothy Horstmann, a well known virologist at Yale University, was sent to the Soviet Union to evaluate the outcome of the trials. Horstmann writes:

It was clear that the trials had been carefully carried out, and the results were monitored meticulously in the laboratory and in the field. By mid-1960 approximately 100 million persons in the Soviet Union, Czechoslovakia, and East Germany had received the Sabin strains. Of great importance was the demonstration that the vaccine was safe, not only for the recipients, but for the large numbers of unvaccinated susceptible who must have been exposed as contacts of vaccines.

The results obtained from these trials in the Soviet Union convinced officials in the US and other countries to carry out clinical trials of OPV. In Japan, Israel, Chile, and other countries, OPV was shown to be highly effective in terminating epidemics of poliomyelitis. In light of these findings, all three of Sabin’s OPV strains were approved for use in the US, and in 1961-62 they replaced IPV for routine immunization against poliomyelitis.

As soon as OPV was used in mass immunizations in the US, cases of vaccine-associated paralysis were described. Initially Sabin decried these findings, arguing that temporal association of paralysis with vaccine administration was not sufficient to implicate OPV. He suggested that the observed paralysis was caused by wild-type viruses, not his vaccine strains.

A breakthrough in our understanding of vaccine-associated paralysis came in the early 1980s when the recently developed DNA sequencing methods were used to determine the nucleotide sequences of the genomes of the Sabin type 3 vaccine, the neurovirulent virus from which it was derived, and a virus isolated from a child who had developed paralysis after administration of OPV. The results enumerated for the first time the mutations that distinguish the Sabin vaccine from its neurovirulent parent. More importantly, the genome sequence of the vaccine-associated isolate proved that it was derived from the Sabin vaccine and was not a wild-type poliovirus.

We now understand that every recipient of OPV excretes, within a few days, viruses that are more neurovirulent that the vaccine strains. This evolution occurs because during replication of the OPV strains in the human intestine, the viral genome undergoes mutation and recombination that eliminate the attenuating mutations that Sabin so carefully selected by passage in different hosts.

From 1961 to 1989 there were an average of 9 cases (range, 1-25 cases) of vaccine-associated paralytic poliomyelitis (VAPP) in the United States, in vaccine recipients or their contacts, or 1 VAPP case per 2.9 million doses of OPV distributed (illustrated). Given this serious side effect, the use of OPV was evaluated several times by the Institute of Medicine, the Centers for Disease Control and Prevention, and the Advisory Committee on Immunization Practices. Each time it was decided that the risks associated with the use of OPV justified the cases of VAPP. It was believed that a switch to IPV would lead to outbreaks of poliomyelitis, because: OPV was better than IPV at protecting non-immunized recipients; the need to inject IPV would lead to reduced compliance; and IPV was known to induce less protective mucosal immunity than OPV.

After the WHO began its poliovirus eradication initiative in 1988, the risk of poliovirus importation into the US slowly decreased until it became very difficult to justify routine use of OPV. In 1996 the Advisory Committee on Immunization Practices decided that the US would transition to IPV and by 2000 IPV had replaced OPV for the routine prevention of poliomyelitis. As a consequence VAPP has been eliminated from the US.

OPV continues to be used in mass immunization campaigns for the WHO poliovirus eradication program, because it is effective at eliminating wild polioviruses, and is easy to administer. A consequence is that neurovirulent vaccine-derived polioviruses (VDPV) are excreted by immunized children. These VDPVs have caused outbreaks of poliomyelitis in areas where immunization coverage has dropped. Because VDPVs constitute a threat to the eradication campaign, WHO has recommended a global transition to IPV. Once OPV use is eliminated, careful environmental surveillance must be continued to ensure that VDPVs are no longer present before immunization ceases, a goal after eradication of poliomyelitis.

As a virologist working on poliovirus neurovirulence, I have followed the vaccine story since I joined the field in 1979. I have never understood why no cases of VAPP were observed in the huge OPV trials carried out in the Soviet Union. Had VAPP been identified in these trials, OPV might not have been licensed in the US. Global use of OPV has led to near global elimination of paralytic poliomyelitis. Would the exclusive use of IPV have brought us to the same point, without the unfortunate cases of vaccine-associated paralysis? I’m not sure we will ever know the answer.

Update: As recently as 1997 DA Henderson, architect of smallpox eradication, argued that developed countries should not use IPV, because it ‘implies accepting the potential of substantial penalties while reducing but not eliminating, an already extremely small risk of vaccine-associated paralytic illness’.

Shedding poliovirus for 28 years

Glass PoliovirusAn immunodeficient individual has been excreting poliovirus in his stool for 28 years. Such chronic excreters pose a threat to the poliovirus eradication program.

Since its inception in 1988 by the World Health Organization, the poliovirus eradication program has relied on the use of the infectious, attenuated vaccine strains produced by Albert Sabin. These viruses are taken orally, replicate in the intestine, and induce protective immunity. During replication in the gut, the Sabin strains lose the mutations that prevent them from causing paralysis. Nearly every individual who receives the Sabin vaccine strains excretes so-called vaccine-derived polioviruses (VDPVs) which are known to have caused outbreaks of poliomyelitis in under-immunized populations.

Immunocompromised individuals who produce very low levels of antibodies (a condition called agammaglobulinemia) are known to excrete VDPVs for very long periods of time – years, compared with months in healthy individuals. Seventy-three such cases have been described since 1962. These individuals receive the Sabin vaccine in the first year of life, before they are known to have an immunodeficiency, at which time they must receive antibodies to prevent them from acquiring fatal infections.

The most recently described immunocompromised patient was found to excrete poliovirus type 2 vaccine for 28 years (the time period is determined by combining the known rate of change in the poliovirus genome with sequence data on viruses obtained from the patient).  The VDPV is neurovirulent (causes paralysis in a mouse model), antigenically drifted, and excreted in the stool at high levels.

Because the polio eradication plan calls for cessation of vaccination at some future time, these immunocompromised poliovirus shedders pose a threat to future unimmunized individuals. The global number of such patients is unknown, and there is no available therapy to treat them – administration of antibodies does not clear the infection. The development of antivirals that could eliminate the chronic poliovirus infection is clearly needed (and ongoing). It will also be necessary to conduct environmental surveillance for the presence of VDPVs – they can be identified by properties that distinguish them from VDPVs produced by immunocompetent vaccine recipients.

While the WHO eradication plan now includes a shift to using inactivated (Salk) poliovaccine, this strategy would not impact the existing immunocompromised poliovirus shedders. Should a VDPV from these individuals cause an outbreak of polio in the post-vaccine era, it will be necessary to control the outbreak with Salk vaccine, or an infectious poliovirus vaccine that cannot revert to virulence during replication in the intestine. Polioviruses with a recoded genome are candidates for the latter type of vaccine.

Image credit: Jason Roberts

TWiV 351: The dengue code

On episode #351 of the science show This Week in Virology, the Masters of the ScienTWIVic Universe discuss a novel poxvirus isolate from an immunosuppressed patient, H1N1 and the gain-of-function debate, and attenuation of dengue virus by recoding the genome.

You can find TWiV #351 at www.microbe.tv/twiv.

Vaccine-associated poliomyelitis in Pakistan

Poliovirus by Jason RobertsAn outbreak of ten cases of poliomyelitis caused by circulating vaccine-derivied poliovirus type 2 (cVDPV2) is ongoing in Pakistan, centered in the Kila Abdulla/Pishin area of Baluchistan. The same virus strain has spread to the neighboring Kandahar province in Afghanistan, where two paralytic cases have been reported. Vaccine-derived poliomyelitis is a well-known consequence of immunization with the Sabin poliovirus vaccine.

There are three serotypes of poliovirus, each of which causes poliomyelitis. The three vaccine strains developed by Albert Sabin (OPV, oral poliovirus vaccine) contain mutations which prevent them from causing paralytic disease. When the vaccine is taken orally, the viruses replicate in the intestine, and immunity to infection develops. While replicating in the intestinal tract, the vaccine viruses undergo genetic changes. As a consequence, the OPV recipients excrete neurovirulent polioviruses. These so-called vaccine-derived polioviruses (VDPV) can cause poliomyelitis in the recipient of the vaccine or in a contact. During the years that the Sabin poliovirus vaccines were used in the US, cases of poliomyelitis caused by VDPV occurred at a rate of about 1 per 1.4 million vaccine doses, or 7-8 per year. Once the disease was eradicated from the US in 1979, the only cases of polio were caused by VDPVs. For this reason the US switched to the Salk (inactivated) poliovirus vaccine in 2000.

Because VDPVs are excreted in the feces, they can spread in communities. These circulating VDPVs, or cVDPVs, can cause outbreaks of poliomyelitis in under-immunized populations. Examples include outbreaks of poliomyelitis in an Amish community and in Nigeria in 2009 caused by cVDPV2. Nigeria employed trivalent OPV before 2003, the year that this country began a boycott of polio immunization. Because type 2 poliovirus had been eradicated from the globe in 1999, when immunization in Nigeria resumed in 2004, monovalent types 1 and 3 vaccine were used. The source of the VDPV type 2 in Nigeria was the trivalent vaccine used before 2003.

For many years the vaccine used by WHO in the global eradication effort was a trivalent preparation comprising all three serotypes. When type 2 poliovirus was eliminated, many countries began immunizing only against types 1 and 3 poliovirus. As a consequence of this immunization strategy, population immunity to type 2 poliovirus declined. This has likely lead to the emergence of cVDPV2 in Pakistan, together with poor routine immunization coverage.

The resurrection of poliovirus type 2 highlights the difficulties in eradicating a pathogen using a vaccine that can readily mutate to cause the disease that it is designed to prevent. As wild type polioviruses are eliminated, the only remaining polio will be caused by the vaccine. If immunization is then stopped, as planned by WHO, there will likely be outbreaks of polio caused by cVDPV of all three serotypes. The solution to this conundrum is to switch to the inactivated vaccine until cVDPVs disappear from the planet.

Exacerbating the polio situation in Pakistan was the murder in the past week of nine immunization workers in several provinces. The Taliban, which carried out the executions, accused them of being spies. This accusation originates from the CIA operation in 2011 in which a Pakistani doctor ran an immunization program in Abbottabad in an attempt to obtain DNA samples from the Bin Laden family. As a result of this violence, immunization campaigns in Balochistan have been suspended. Coupled with the previous refusal of many parents to have their children immunized, this action makes it likely that poliovirus will spread more extensively in the country, making eradication even more difficult.

Poliovirus image courtesy of Jason Roberts.

Poliovirus vaccine safety

Albert SabinThe contamination of the rotavirus vaccine Rotarix with porcine circovirus 1 DNA was revealed by deep sequencing. The same technique was also used to demonstrate that oral poliovirus vaccine does not contain viruses that can cause poliomyelitis.

The oral poliovirus vaccine strains developed by Albert Sabin (pictured) were licensed in the United States in 1962, and over the next 37 years immunization with these vaccines lead to the eradication of poliomyelitis in this country. During that period, the vaccine was responsible for 5-10 cases of poliomyelitis each year, either in recipients of the vaccine or in their contacts. Some of these individuals have sued the manufacturers of the vaccine, claiming that they made a defective product.

OPV contains three different poliovirus strains which were selected by Sabin because they do not cause poliomyelitis. We call such vaccine strains avirulent or attenuated. The mutations in the genetic information of the virus that prevent the development of paralysis have been identified. Unfortunately, these mutations are unstable. After oral administration, OPV replicates in the intestinal tract. During this phase the vaccine viruses undergo genetic change and eventually lose the mutations that made them avirulent. As a consequence, nearly every infant who receives OPV sheds in the feces polioviruses that are significantly more neurovirulent than those that were ingested.

Vaccine-associated poliomyelitis is caused by vaccine revertants that accumulate in the alimentary tract of immunized individuals. These neurovirulent viruses arise not because the vaccine is improperly prepared, but as a consequence of mutation during replication in the intestine. Proving this point to lay juries has been difficult. Now deep sequencing of poliovirus vaccine can show whether or not vaccine preparations are contaminated with neurovirulent viruses.

Deep sequence analysis of OPV manufactured by Bharat Biotech was done to detect mutations associated with neurovirulence. There are four mutations in the genome of type 1, two in the genome of type 2, and three in the genome of type 3 that are important for the attenuated property of the vaccine. The base present at each of these positions in the neurovirulent wild type viruses, and in the vaccine strains, is shown in the table.

Determinants of attenuation

The results of sequence analysis show that the Bharat vaccine does not contain any of the ‘wild type’ bases at these nine positions. Any vaccine-associated poliomyelitis associated with this vaccine is not a consequence of faulty production, but the fact that vaccine strains mutate during replication in the human gut.

There have been many lawsuits involving vaccine-associated poliomyelitis in which plaintiffs claim that the OPV was incorrectly manufactured, leading to a product of unacceptably high neurovirulence. Deep sequencing analysis of these lots of vaccine could have resolved this claim in a way that a lay jury could understand.