Blocking virus infection with soluble cell receptors

poliovirus + receptorWe recently discussed the development of a soluble receptor for HIV-1 that provides broad and effective protection against infection of cells and of nonhuman primates. Twenty-five years ago my laboratory published a paper which concluded that using soluble receptors to block virus infection might not be a good idea. In the first paragraph of that paper we wrote:

…it has been proposed that soluble cell receptors might be effective antiviral therapeutics. It has been suggested that mutants resistant to the antiviral effects of soluble receptors would not arise, because mutations that abrogate binding to receptors would be lethal.

We had previously shown that the cell receptor for poliovirus, CD155, produced in a soluble form, would bind to poliovirus (pictured – the very image from the banner of this blog), blocking viral infection. We then found that it was relatively easy to select for soluble receptor resistant (srr) virus mutants. These viruses still enter cells by binding to CD155, but the affinity of virus for the receptor is reduced. Poliovirus srr mutants replicate normally in cell cultures, and cause paralysis in a mouse model for poliomyelitis. We speculated that receptor binding might not be a rate-limiting step in viral infection, and short of  abolishing binding, the virus can tolerate a wide range of binding capabilities.

The amino acid changes that cause the srr phenotype map to both the exterior and the interior of the viral capsid. The changes on the virion surface are likely to directly interact with the cell receptor. Changes in the interior of the virus particle may be involved in receptor-mediated conformational transitions that are believed to be essential steps in viral entry.

When this work was done, clinical trials of soluble CD4 for HIV-1 infection were under way. We believed that our findings did not support the use of soluble receptors as antivirals, which we clearly stated in the last sentence of the paper:

These findings temper the use of soluble receptors as antiviral compounds.

HIV-1 mutants resistant to neutralization with soluble CD4 were subsequently isolated, and the compound was never approved to treat HIV-1 infection in humans for this and other reasons, including low affinity for the viral glycoprotein, enhancement of infection, and problems associated with using a protein as a therapeutic.

Recently a new soluble CD4 was produced which also includes the viral binding site for a second cell receptor, CCR5. This molecule overcomes many of the issues inherent in the original soluble CD4. It provides broad protection against a wide range of HIV-1 strains, and when delivered via an adenovirus-associated virus vector, protects nonhuman primates from infection. This delivery method circumvents the issues inherent in using a protein as an antiviral drug. Because this protein blocks both receptor binding sites on the viral envelope glycoprotein, it might be more difficult for viruses to emerge that are resistant to neutralization. The authors speculate that such mutants might not be efficiently transmitted among hosts due to defects in cell entry. Given the promising results with this antiviral compound, experiments to test this speculation are certainly welcome.

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.

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.

HIV gets the zinc finger

zinc finger nucleaseBecause all animal viruses initiate infection by binding to a receptor on the cell surface, this step has long been considered a prime target for antiviral therapy. Unfortunately, drugs that block virus attachment to cells have never shown much promise. Another approach, which is to ablate the receptor from the cell surface, is also problematic because these molecules have essential cellular functions. Removing one of the receptors for human immunodeficiency virus type 1 might be an exception.

HIV-1 must interact with two cell surface proteins to initiate infection: a T lymphocyte protein called CD4, and a second receptor, which can be one of two molecules called CCR5 or CXCR4. For many years it has been known that humans can survive without the CCR5 protein: from 4-16% of people of European descent carry the ccr5-delta32 mutation, that prevents the protein from reaching the cell surface. Individuals who are homozygous for ccr5-delta32 (the mutation is present in both copies of the gene) are resistant to HIV infection. Because the vast majority of HIV viruses that are transmitted are those that require CCR5 for cell entry, absence of the protein on the cell surface confers resistance to infection.

The key role of CCR5 in HIV infection in humans was further confirmed when an AIDS patient was given a bone marrow transplant from a donor with the ccr5-delta32 mutation. The patient has been free of HIV for years despite not taking anti-retroviral drugs.

These findings suggest that one possible therapy for AIDS would be to disrupt the ccr5 gene in patient lymphocytes. The development of gene-targeting technologies has brought this approach closer to reality. One approach uses zinc finger nucleases, which are artificial proteins made by joining a protein that can specifically bind DNA with an enzyme that can cleave DNA. A zinc finger nuclease can be designed, for example, to specifically cut within the ccr5 gene. When the cell tries to repair the cut, the gene may be damaged so that the CCR5 protein is no longer made (illustrated).

This approach works: when CD4 T lymphocytes are removed from humans, cultured, and treated with a ccr5 zinc finger nuclease, they become resistant to HIV infection. We discussed this experiment on episode #144 of This Week in Virology.

The next step has now been done: to remove CD4 T lymphocytes from HIV positive donors, treat the cells with the ccr5 zinc finger nuclease (delivered using an adenovirus vector), and infuse the cells back into the patients (each person receives his or her own modified cells). Half of the donors were removed from anti-retroviral therapy, and then the levels of HIV, and CD4 lymphocytes, were measured over the next 250 days.

The result were encouraging: not only were the infusions safe, but the overall levels of CD4 lymphocytes increased, and a good fraction of these had modified ccr5 genes. The initial rise of HIV viremia after interruption of treatment was followed by a decline in virus load. These results show that the CD4 T lymphocytes with modified ccr5 were able to expand in the recipients, and survived better than the unaltered lymphocytes, probably because they were at least partially resistant to HIV infection.

This important clinical trial is only the beginning of a new approach to HIV therapy, and several substantial problems still remain to be solved. Both copies of the ccr5 gene were modified in only 33% of the CD4 T lymphocytes; the remaining cells can still be infected by HIV, albeit less efficiently. New approaches are needed to disrupt both copies of the ccr5 gene in most of the T lymphocytes.

Another issue is that the modified T cells can proliferate for a long time, but not indefinitely. As these cells divide from a limited number of infused cells, they will not have the broad repertoire needed to fight pathogens. T cells are also known to become “exhausted”: they eventually lose their protective functions. Patients given modified lymphocytes still harbor a pool of long-lived T cells which contain HIV DNA. These cells will likely always be present and could give rise to viremia. CD4 T lymphocytes with normal levels of ccr5 protein will always be produced, serving as potential hosts for HIV replication. Modifying stem cells so that they do not produce CCR5 is one long-term solution, but more difficult and dangerous for the patient.

Despite these drawbacks, it is amazing that we can now remove cells from patients, modify their genes, and place them back in patients with little harm and some clear benefit. This is a complicated set of procedures, made even more difficult because humans are involved. It’s truly a landmark clinical trial.

Antimicrobial peptides induced by herpesvirus enhance HIV-1 infection

Langerhans cellsThe risk of being infected with human immunodeficiency virus type 1 (HIV-1) is substantially enhanced in individuals with other sexually transmitted diseases. For example, infection with herpes simplex virus type 2 (HSV-2) increases the risk ratio of acquiring HIV from 2 to 4. Explanations for this increased risk include direct inoculation of HIV-1 into the blood through genital ulcers, and the induction of inflammatory cells by HSV-2 which act as sites of replication for HIV-1. The results of infections carried out in cell culture suggest a biological mechanism for the enhancement of HIV-1 infection by HSV-2.

Langerhans cells (LC) are believed to one of the first cells in which HIV-1 replicates after sexual exposure. LCs are dendritic cells which patrol the mucosal epithelium, taking up and processing antigens and presenting them to T cells in the lymph nodes. These cells express the HIV-1 receptors CD4 and CCR5, but not CXCR4, and can therefore be infected with CCR5-tropic* but not CXCR4-tropic HIV-1. Individuals who do not express CCR5 are resistant to HIV infection. For these and other reasons CCR5-tropic HIV-1 viruses are believed to be ones that transmit infection from one individual to another.

In human skin explant cultures, which contain LCs, co-infection with HSV-2 substantially increased the number of HIV-1 cells. This observation could not be explained by co-infection of individual cells because very few of these were observed in the cultures. When applied to fresh cells, the supernatant of cultures infected with HSV-2 also stimulated the number of HIV-1 infected LCs. These observations suggested that HSV-2 infection stimulates the production of one or more substances from infected cells which in turn improve HIV-1 infection.

Human epithelial and epidermal cells are known to produce antimicrobial peptides such as defensins and cathelicidin. These are short, evolutionarily conserved peptides that inhibit the growth of bacteria, viruses, and fungi. HSV-2 infected keratinocytes were found to produce a number of antimicrobial peptides, but the most important one is called LL-37. This peptide enhanced the expression of HIV-1 receptors CD4 and CCR5 on LCs, leading to increased susceptibility of the cells to HIV-1. Removing LL-37 from the supernatant of HSV-2 infected cells reduces the ability of the medium to stimulate susceptibility to HIV-1.

These findings provide a plausible mechanism by which HIV-1 infection is enhanced by HSV-2. When HSV-2 infects the genital mucosa, the epithelial cells produce LL-37. This antimicrobial peptide enhances the production of CD4 and CCR5 on LCs, allowing more efficient infection by HIV-1. This mechanism is supported by the observation that elevated levels of LL-37 correlate with HIV-1 infection in sex workers.

I wonder why antimicrobial peptides up-regulate CD4 and CCR5. In addition to their antimicrobial properties, the cathelicidins possess chemotactic, immunostimulatory, and immunomodulatory effects, and the upregulation of CD4 and CCR5 are likely part of these activities.

These are exciting findings, and if they are further correlated in humans, they might lead to novel ways of interfering with HIV-1 infection, such as by antagonizing LL-37.

*CCR5 and CXCR4-tropic refer to HIV-1 virions that bind to chemokine receptors CCR5 or CXCR4, respectively, in addition to CD4, to initiate infection.

Did smallpox lead to HIV-1 resistance?

10661_loresThe 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 observation was the basis for giving an AIDS patient a bone marrow transplant from a donor with the delta 32 mutation: his lymphocytes became resistant to HIV-1 infection, and he has been free of virus for over two years.

Approximately 10% of the human population carries the CCR5 delta 32 deletion (although it is rare in Africans and Asians). But HIV-1 is a recent invader of humans – it is believed to have crossed from chimpanzees around 1930. This length of time is far too short to have provided sufficient selection pressure to retain the CCR5 delta 32 mutation in humans. Instead, the selection pressure may have been provided by another human viral infection: smallpox.

Myxoma virus, a member of the poxvirus family, causes lethal disease in rabbits. Mouse cells that cannot be infected by this virus can be made susceptible to infection by expression of genes encoding several chemokine receptors, including CCR5. Furthermore, myxoma virus infection of CCR5-expressing mouse cells can be blocked with antibody to CCR5 or RANTES, its natural ligand. These observations indicate that CCR5 can serve as an entry receptor for myxoma virus.

Smallpox, a virus in the same family as myxoma virus, has been infecting humans for thousands of years – the earliest outbreaks are believed to have occurred before 1000 AD. The receptor for smallpox virus is not known, but if it is CCR5, then smallpox is the leading candidate for the selective pressure responsible for fixation of the CCR5 delta 32 HIV-1 resistance allele in modern Caucasians. 

A. S. Lalani (1999). Use of Chemokine Receptors by Poxviruses Science, 286 (5446), 1968-1971 DOI: 10.1126/science.286.5446.1968