On episode #301 of the science show This Week in Virology, Vincent travels to the International Congress of Virology in Montreal and speaks with Carla Saleh and Curtis Suttle about their work on RNA interference and antiviral defense in fruit flies, and viruses in the sea, the greatest biodiversity on Earth.

You can find TWiV #301 at www.twiv.tv.

The Berlin patient

6 September 2014

HIV binding CD4 and ccrSince the beginning of the AIDS epidemic, an estimated 75 million people have been infected with HIV. Only one person, Timothy Ray Brown, has ever been cured of infection.

Brown was diagnosed with HIV while living in Berlin in 1995, and was treated with anti-retroviral drugs for more than ten years. In 2007 he was diagnosed with acute myeloid leukemia. When the disease did not respond to chemotherapy, Brown underwent stem cell transplantation, which involves treatment with cytotoxic drugs and whole-body irradiation to destroy leukemic and immune cells, followed by administration of donor stem cells to restore the immune system. When his leukemia relapsed, Brown was subjected to a second stem cell transplant.

The entry of HIV-1 into lymphocytes requires two cellular proteins, the receptor CD4, and a co-receptor, either CXCR4 or CCR5. Individuals who carry a mutation in the gene encoding CCR5, called delta 32, are resistant to HIV-1 infection. This information prompted Brown’s Berlin physician to screen 62 individuals to identify a stem cell donor who carried a homozygous CCR5∆32 mutation. Peripheral blood stem cells from the same donor were used for both transplants. 

Despite enduring complications and undergoing two transplants, Brown’s treatment was a success: he was cured both of his leukemia and HIV infection. Even though he had stopped taking antiviral drugs, there was no evidence of the virus in his blood following his treatment, and his immune system gradually recovered. Follow-up studies in 2011, including biopsies from his brain, intestine, and other organs, showed no signs of HIV RNA or DNA, and also provided evidence for the replacement of long-lived host tissue cells with donor-derived cells. Today Brown remains HIV-1 free.

Although Brown’s cure is somewhat of a medical miracle, and by no means a practical road map for treating AIDS, the example of the Berlin patient has galvanized research efforts and continues to inspire hope that a simpler and more general cure for infection may someday be achieved. Clinical trials have been conducted to test a variety of strategies in which CD4+ T or stem cells are obtained from a patient, the CCR5 gene is either mutated or its translation blocked by RNA interference, and then the resulting virus-resistant cells are returned to the patient. In one case zinc finger nucleases were used to delete the CCR5 gene in a patient’s cells, a procedure that we discussed in TWiV #278.

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Recording together for the first time, the hosts of the science show This Week in Virology celebrate their 300th recording at the American Society for Microbiology headquarters in Washington, DC, where Vincent  speaks with Dickson, Alan, Rich, and Kathy about their careers in science.

You can find TWiV #300 at www.twiv.tv.

Antibodies bound to poliovirus.

Antibodies (purple) bound to poliovirus. Image credit: Jason Roberts

Antigenic variation is a hallmark of influenza virus that allows the virus to evade host defenses. Consequently influenza vaccines need to be reformulated frequently to keep up with changing viruses. In contrast, antigenic variation is not a hallmark of poliovirus – the same poliovirus vaccines have been used for nearly 60 years to control infections by this virus. An exception is a poliovirus type 1 that caused a 2010 outbreak in the Republic of Congo.

The 2010 outbreak (445 paralytic cases) was unusual because the case fatality ratio of 47% was higher than typically observed (usually less than 10% of patients with confirmed disease die). The first clue that something was different in this outbreak was the finding that sera from some of the fatal cases failed to effectively block (neutralize) infection of cells by the strain of poliovirus isolated during this outbreak (the strain is called PV-RC2010). The same sera effectively neutralized the three Sabin vaccine viruses as well as wild type 1 polioviruses isolated from previous outbreaks. Therefore gaps in vaccination coverage were solely not responsible for this outbreak.

Examination of the nucleotide sequence of the genome of type I polioviruses isolated from 12 fatal cases revealed two amino acid changes within a site on surface of the viral capsid that is bound by neutralizing antibodies (illustration). The sequence of this site, called 2a, was changed from ser-ala-ala-leu to pro-ala-asp-leu. This particular combination of amino acid substitutions has never been seen before in poliovirus. Virus PV-RC2010, which also contains these two amino acid mutations, is completely resistant to neutralization with monoclonal antibodies that recognize antigenic site 2 (monoclonal antibodies recognize a single epitope, as opposed polyclonal antibodies which is a mixture of antibodies that recognize many epitopes. The antibodies in serum are typically polyclonal).

Poliovirus neutralization titers were determined using sera from Gabonese and German individuals who had been immunized with Sabin vaccine. These sera effectively neutralized the type I strain of Sabin poliovirus, as well as type 1 polioviruses isolated from recent outbreaks. However the sera had substantially lower neutralization activity against PV-RC2010. From 15-29% of these individuals would be considered not to be protected from infection with this strain.

Nucleotide sequence analysis of PV-RC2010 reveals that it is related to a poliovirus strain isolated in Angola in 2009, the year before the Republic of Congo outbreak. The Angolan virus had just one of the two amino acid changes in antigenic site 2a found in PV-RC2010.

It is possible that the relative resistance of the polioviruses to antibody neutralization might have been an important contributor to the high virulence observed during the Republic of Congo outbreak. The reduced ability of serum antibodies to neutralize virus would have lead to higher virus in the blood and a greater chance of entering the central nervous system. Another factor could also be that many of the cases of poliomyelitis were in adults, in which the disease is known to be more severe.

An important question is whether poliovirus strains such as PV-RC2010 pose a global threat. Typically the fitness of antigenically variant viruses is not the same as wild type, and therefore such viruses are not likely to spread in well immunized populations. Today some parts of the world have incomplete poliovirus immunization coverage, which together with the reduced circulation of wild type polioviruses leads to reduced population immunity. Such a situation could lead to the evolution of antigenic variants. This situation occurred in Finland in 1984, when an outbreak caused by type 3 poliovirus took place. The responsible strains were antigenic variants that evolved due to use of a sub-optimal poliovirus vaccine in that country.

The poliovirus outbreaks in the Republic of Congo and Finland were stopped by immunization with poliovirus vaccines, which boosted the population immunity. These experiences show that poliovirus antigenic variants such as PV-RC2010 will not cause outbreaks as long as we continue extensive immunization with poliovirus vaccines, coupled with environmental and clinical testing for the presence of such viruses.

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On episode #299 of the science show This Week in VirologyVincent visits the Rocky Mountain Laboratories in Hamilton, Montana and speaks with Marshall Bloom, Sonja Best, and Byron Caughey about their work on tick-born flaviviruses, innate immunity, and prion diseases.

You can find TWiV #299 at www.twiv.tv.

Many people have a new awareness of the disease known as amyotrophic lateral sclerosis, or ALS, thanks to the Ice Bucket Challenge initiated by the ALS Association. Fewer might know that retroviruses have been proposed to play a role in the development of the disease.

I previously summarized a 2008 paper on ALS in a piece called Retroviruses and amyotrophic lateral sclerosisSera from some ALS patients had previously been shown to contain elevated levels of reverse transcriptase, an enzyme found in retrovirus particles. In the 2008 paper, RNAs encoding this enzyme were reported in the brains of ALS patients, and their origin appears to be the human endogenous retrovirus HERV-K.

The progress made in understanding the relationship of endogenous retroviruses with ALS is summarized in a review published in August of 2014 entitled Retroviruses and amyotrophic lateral sclerosis (the paper is open access). The authors conclude:

A comprehensive study of the expression or reactivation of endogenous retroviral elements in ALS has not yet been undertaken. The literature on HERV-W involvement in ALS is difficult to interpret. Two independent reports, however, have shown increased HERV-K expression in both serum and brain tissue in ALS patients. It remains unknown if HERV-K expression is an epiphenomenon or plays a pathophysiological role in the disease.

I am pleased to participate in the Ice Bucket Challenge to help raise awareness of ALS and raise money to work on the disease.

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TWiV 298: MV-NIS de myelo

17 August 2014

On episode #298 of the science show This Week in Virology, the TWiV gang answers follow-up questions about the Ebola virus outbreak in West Africa, then discuss treatment of  disseminated multiple myeloma with oncolytic measles virus.

You can find TWiV #298 at www.twiv.tv.

TWiV 300This Week in Virology, the podcast about viruses – the kind that may or may not make you sick, celebrates its 300th episode on Tuesday, August 26, 2014 with a live recording at the Washington, DC headquarters of the American Society for Microbiology. This special episode will be part of the ‘Microbes after Hours’ series, and will feature the TWiV hosts Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler recording together in person for the first time.

TWiV 300 will be live-streamed, but if you live in the Washington, DC area, you are welcome to join us and watch the episode in person. We have a limited number of seats available on a first come, first serve basis. Click the RSVP link below to register.

Date: Tuesday, August 26, 2014

Reception from 6-7 PM at ASM Headquarters, 1752 N Street, N.W. Washington, D.C. 20036-2904

TWiV 300th Episode live from 7-8 PM RSVP required to attend.

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On episode #297 of the science show This Week in Virology, the TWiVites present an all-ebolavirus episode, tackling virology, epidemiology, and approaches to prevention and cure that are in the pipeline.

You can find TWiV #297 at www.twiv.tv.

guinea-liberia-sierra-leone-2014As the epidemic of Zaire ebolavirus in Western Africa continues (1,779 cases and 961 deaths in four countries), many are questioning why there are no means of preventing or stopping infection. In the past two decades there has been substantial research into developing and testing active and passive vaccines and antiviral drugs, although none have yet been licensed for use in humans.

Using antibodies to treat infection with ebolaviruses with antibodies is probably the best known therapy, because it was used to treat a two Americans who were infected while working in Liberia. They received a mixture of three monoclonal antibodies (called ZMapp) which had been previously shown to block infection of cells with ebolaviruses, and prevent lethal infection of non-human primates when given within 24-48 hours after infection. These are mouse monoclonal antibodies that have been ‘humanized’ so that when given to people they do not induce an antibody response against the antibodies. Humanization involves changing the amino acids of the antibody molecule from mouse to human, except in the part of the antibody that binds antigen. The antibodies are then synthesized in tobacco plants and purified. Administering anti-viral antibodies to patients, also called passive immunization, was done long before vaccines were available. Serum from patients who had recovered from a particular disease would be given to others who had recently been infected, in order to prevent disease. Such therapy was used to save the life of virologist Jordi Casals, who had become infected with Lassa virus while isolating the virus from the blood of a patient, Penny Pinneo. The serum administered to Casals was obtained from Pinneo, who had recovered from the infection. The American doctor infected with Zaire ebolavirus while working in Liberia was also given serum from a boy who had recovered from infection.

As ZMapp has not yet been subjected to human clinical trials to determine its safety and efficacy, its use in an infected human is considered unusual. A phase I clinical trial needs to be done to ensure that the preparation of monoclonal antibodies is safe in humans. Determining whether monoclonal antibody therapy for ebolavirus infection is effective is more difficult. Such testing could only be done during an outbreak, during which it would not be ethical to withhold treatment from the control group. Nevertheless it is clear that such mixtures of monoclonal anti-viral antibodies could potentially save many lives during outbreaks.

While passive immunization has value in saving lives, its protection is temporary: the antibodies given to patients do not endure. A better approach is immunization, which not only induces anti-viral antibodies, but creates immune memory, so that subsequent infections are accompanied by another round of antibody production. The catch is that it takes about two weeks after immunization for antibodies to reach sufficient protective levels. Nevertheless, a vaccine would likely have had substantial impact on the current outbreak, which began in March 2014 and has continued for 5 months.

A number of experimental vaccines against ebolaviruses are in development. In one approach, the glycoprotein of vesicular stomatitis virus is replaced with the corresponding protein of different ebolaviruses. These vaccines protect non-human primates from lethal infection. A similar approach using an attenuated rabies virus to deliver the ebolavirus glycoprotein also protected non-human primates from infection, as did immunization with an adenovirus encoding the ebolavirus glycoprotein.  This vaccine candidate has been shown to be safe and immunogenic in phase I clinical trials. Another vaccine approach entails production of the ebolavirus glycoprotein in E. coli. Immunization of mice with the purified protein leads to the production of neutralizing antibodies. Because protein-based vaccines do not replicate, the immune response may need to be boosted by using an adjuvant that stimulates the innate immune system and leads to better antibody production. A double-stranded RNA adjuvant has been shown to augment the immune response against a non-infections, virus-like particle vaccine containing the Ebola virus glycoprotein but not the viral genome.

Antivirals certainly have a place in control of viral disease, and a number of promising candidates to control infection with ebolaviruses have been developed. One is a nucleoside analog which is incorporated into RNA by the viral RNA polymerase and leads to chain termination. It blocks replication of ebolaviruses in culture cells, and protects mice and nonhuman primates from lethal infection. This compound, called BCX4430, is a broad spectrum antiviral that inhibits the replication of not only members of the Filoviridae, but also Arenaviridae, Bunyaviridae, Orthomyxoviridae, Picornaviridae, Paramyxoviridae, Flaviviridae, Coronaviridae. Another inhibitor of viral RNA synthesis is favipiravir, which has the advantage of being in late stage clinical development for the treatment of influenza. This compound inhibits replication of ebolaviruses in cultured cells and reduces disease severity and mortality in a mouse model of disease.

It is likely that the extent of the current outbreak of Ebola virus disease, the largest to date, will provide impetus to move some of these treatments into human trials. But consider that all the research on active and passive vaccines and antivirals for ebolaviruses required work in BSL-4 laboratories. Those who call for the shuttering of BSl-4 laboratories need to take note and move away from their unrealistic and unreasonable position.

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