TWiV 320: Retroviruses and cranberries

On episode #320 of the science show This Week in Virology, Vincent speaks with John Coffin about his career studying retroviruses, including working with Howard Temin, endogenous retroviruses, XMRV, chronic fatigue syndrome and prostate cancer, HIV/AIDS, and his interest in growing cranberries.

You can find TWiV #320 at www.microbe.tv/twiv.

Combination antiviral therapy for hepatitis C

Ledipasvir and SofosbuvirThe Food and Drug Administration has approved the use of a single pill containing two different antiviral drugs for the treatment for hepatitis C. It is the first combination pill approved for the disease, and also the first treatment that does not contain interferon or ribavirin.

The new hepatitis C drug, called Harvoni, is a mixture of the antiviral drugs ledipasvir and sofosbuvir. Ledipasvir (pictured) is an inhibitor of the hepatitis C virus protein NS5A, which has multiple roles in the viral replication cycle that include RNA synthesis and virus particle assembly. The mechanism of NS5A inhibition by ledipasvir is not known. Sofosbuvir is a previously licensed inhibitor that targets the viral RNA-dependent RNA polymerase. It is an analog of the nucleoside uridine, one of the four building blocks of RNA. Sofosbuvir is utilized by the viral RNA polymerase, leading to inhibition of viral RNA synthesis.

The use of single antiviral drugs (monotherapy) to treat RNA virus infections is always problematic because resistance usually arises rapidly. Dual-therapy pills like Harvoni are better, but the best are triple-therapy pills. Triple therapy formulations such as Atripla have been used successfully to treat infections with HIV-1, and presumably there will be mixtures of three antiviral drugs for treating hepatitis C.

Let’s use HIV-1 to illustrate the value of treating infections with multiple antiviral drugs. The HIV-1 viral genome, like that of HCV, is slightly less than 10,000 bases long. Assume that one mutation in the viral genome is needed for drug resistance. If the RNA polymerase mutation rate is 1 out of every 10,000 bases synthesized, then each base in the viral genome is substituted in a collection of 10,000 viruses. An HIV-1 infected person can make as many as 10,000,000,000 virus particles each day, so 1010/104 = one million viruses will be produced each day with resistance to one drug.

If we use two antiviral drugs, developing resistance to both occurs in every 104 x 104 = 108 viruses. In this case 1010/108 = 100 viruses will be produced each day with resistance to two drugs.

If we use three antiviral drugs, developing resistance occurs in every 104 x 104 x 104= 1012 viruses, which is more than what is produced each day.

This is why triple antiviral therapy has been so successful for the treatment of AIDS.

And yes, I’m sure someone has tested Sofosbuvir for inhibition of Ebola virus replication.

The Berlin patient

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.

TWiV 293: Virology Down Under

On episode #293 of the science show This Week in VirologyVincent visits Melbourne, Australia and speaks with Melissa, Alex, Gilda, and Paul about their work on HIV infection of the central nervous system, West Nile virus, microbicides for HIV, and the Koala retrovirus.

You can find TWiV #293 at www.microbe.tv/twiv.

TWiV 278: Flushing HIV down the zinc

On episode #278 of the science show This Week in Virology, Vincent, Dickson, Alan, and Kathy discuss disruption of the ccr5 gene in lymphocytes of patients infected with HIV-1.

You can find TWiV #278 at www.microbe.tv/twiv.

TWIV 266: A pathogenic vicious cycle

On episode #266 of the science show This Week in Virology, Vincent, Alan, and Kathy discuss finding viruses in outer space, varying results obtained from personal genetic testing, and depletion of CD4 cells during HIV infection by pyroptosis.

You can find TWiV #266 at www.microbe.tv/twiv.

TWiV 260: Badgers go viral

On episode #260 of the science show This Week in Virology, Vincent visits the University of Wisconsin, Madison and speaks with Tom, Tony, and David about their work on virus discovery at the AIDS Vaccine Research Laboratory.

You can find TWiV #260 at www.microbe.tv/twiv.

TWiV 244: Back in the CVVR

Episode #244 of the science show This Week in Virology was recorded before an audience at the Beth Israel Deaconess Medical Center, where Vincent and Alan spoke with Dan and Jeff about AIDS vaccines.

You can find TWiV #244 at www.microbe.tv/twiv.

TWiV 237: Paleovirology with Michael Emerman

Episode #237 of the science show This Week in Virology was recorded at the Fred Hutchinson Cancer Research Center in Seattle, WA, where Vincent and Rich met up with Michael to talk about his work on the molecular and evolutionary basis of HIV replication and pathogenesis.

You can find TWiV #237 at www.microbe.tv/twiv.

Hilary Koprowski, 96

Racaniello, Koprowski, KessinVirologist Hilary Koprowski died on 11 April 2013 at the age of 96. His main accomplishments are nicely summarized in the New York Times, but for a more comprehensive overview of his life, I highly recommend his biography Listen to the Music by Roger Vaughan. I did not have many opportunities to interact with Dr. Koprowski, but I did follow his work on poliovirus vaccines and I have a few reminiscences.

In the 1930s Max Theiler had found that propagating yellow fever virus in an unnatural host – the chick embryo – dramatically reduced its capacity to cause disease in humans. Theiler’s work (which garnered him a Nobel Prize) lead to the production of the infectious, attenuated yellow fever vaccine which helped to vastly reduce the global incidence of yellow fever. Koprowski was inspired by Theiler’s work and decided to take a similar approach to developing a poliovirus vaccine – his first efforts involved passage of a type 2 strain of poliovirus in mice and then in cotton rats. After passage in  rodents, the virus did not cause paralysis in monkeys. Koprowski tested the candidate vaccine strain in humans, and ultimately produced two other attenuated poliovirus strains. By the 1960s these attenuated poliovirus vaccine candidates had been tested in millions of humans. However, they were never licensed for use in the US. While Koprowski was carrying out his work, Albert Sabin was also developed attenuated vaccine strains of poliovirus. Both Sabin’s and Koprowski’s strains were tested side by side in a monkey neurovirulence test carried out by Joseph Melnick at Baylor University. Sabin’s virus strains were slightly more attenuated, and in 1961-62 those were selected for licensing in the US. Sabin’s oral poliovirus vaccines (OPV) have been the mainstay of the World Health Organization in its polio eradication campaign.

Koprowski’s polio vaccines were tested by human clinical trials, notably in the former Belgian Congo in 1957-58. It was subsequently suggested that this clinical trial initiated the AIDS pandemic. The idea, first proposed by Tom Curtis (19 March 1992 “The Origin of AIDS: A startling new theory attempts to answer the question, ‘Was it an act of God or an act of man?” Rolling Stone pp. 54–9, 61, 106, 108) and subsequently by Edward Hooper in ‘The River‘, was that Koprowski had propagated the vaccine strains in kidney cell cultures produced from locally captured chimpanzees. If these animals were infected with the precursor of HIV-1, simian immunodeficiency virus (SIV), then the virus might have entered the human population during the polio vaccine trials. This hypothesis was subsequently shown to be incorrect as phylogenetic analysis showed that the main group of HIV-1 viruses, the M group, clearly crossed from chimpanzees to humans in the early 1900s.

A committee was established to investigate the virological aspects of the HIV-polio vaccine controversy, and towards the end of its work I was asked to join. When it was discovered that samples of Koprowski’s polio vaccines were frozen at the Wistar Institute in Philadelphia, it was decided to determine whether these vaccines had been propagated in rhesus monkey or chimpanzee cells.

I was given the job of dividing and coding the samples. I met a representative of the Wistar Institute in the parking lot of a restaurant just off the New Jersey Turnpike, halfway between New York and Philadelphia. He handed me a white styrofoam box, packed with ice, that contained  vials of the Koprowski vaccine. To the uninformed observer, it might have looked like a drug exchange.

CHAT vialsI took the vials back to the lab (see photo), thawed them, separated them into aliquots, and gave each a code. I then returned them to the Wistar in the same way, after a second trip on the New Jersey Turnpike. The samples were sent to three different laboratories where experiments were done to determine the mitochondrial DNA type of the cells in which the viruses had been propagated (although the samples were free of cells, some mitochondrial DNA would still be present from virus induced cell lysis). The results, which have been published (reference one, two), clearly showed that the vaccines had not been grown in chimpanzee cells. I was pleased to have played a small role in this story.

Koprowski autograph

Although I had spoken with Dr. Koprowski several times on the telephone, I did not meet him until 2005 when he presented a talk on the history of rabies in the History of Science series at Columbia University Medical Center. I was his host for that visit, during which I was photographed with Dr. Koprowski and Dr. Rich Kessin, another Columbia professor. We invited Dr. Koprowski to dinner after the seminar but he declined, but he did want to have a drink together. After being warned by his driver not to keep him too late, we walked to a local bar and Dr. Koprowski ordered a Boodles gin martini. The bartender noted that he didn’t receive many calls for that brand. Dr. Koprowski said it was his favorite gin. We talked for a while and then returned Dr. Koprowski to his car for the trip back to Philadelphia. During his visit I had Dr. Koprowski autograph my copy of Listen to the Music (photo). He wrote: “To my friend Vince, with warmest regards, Hilary, 4/14/05.” It was the first and last time I saw him.

I would like to relate one last story which has nothing to do with me, but is irresistible. It takes place in the opening pages of Listen to the Music. Koprowski and his technician Thomas Norton are about to drink an early version of his attenuated polio vaccine. The virus has been passaged in rats and appears to be attenuated in monkeys. On a January day in 1948 Koprowski and Norton are in a laboratory at Lederle Laboratories in Pearl River, NY., where they are blending the brains and spinal cords of rats that had been infected with the vaccine strain virus. They both drink a milliliter of the cold, greasy, mix which flows thickly over their tongues and is difficult to swallow.

Here is the best part:

When he can speak, Norton asks, “Have another?”

“Better not,” Koprowski says. “I’m driving.”