virology blog

About viruses and viral disease

T helper cells

The Esperanza Patient

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by Gertrud U. Rey

There is still no real cure for HIV infection. Only two people have been intentionally and successfully cleared of the virus thus far – the Berlin patient and the London patient. However, both subjects needed dangerous stem cell transplants to replenish their blood stem cells that had been destroyed during chemotherapy regimens needed to treat their HIV-induced blood cancers. In their transplants, doctors used bone marrow cells from a donor who was homozygous for a mutation in the gene encoding the HIV co-receptor CCR5 (CCR5 Δ32/Δ32), because this genotype confers resistance to HIV-1 infection. Such a transplant strategy cannot be realistically applied to most HIV patients.

Recently, a thirty-year-old female resident of Esperanza, Argentina, was declared to be cured of HIV-1 without receiving long-term treatment. The “Esperanza patient” is actually the second individual known to have cleared the infection naturally. The first person, known as the “San Francisco patient,” is a 67-year-old woman who appears to have cleared the virus in the absence of treatment after living with HIV for 28 years. Standard HIV treatment involves a combination of drugs known as antiretroviral therapy (ART), which is very effective at reducing the viral load in the blood of infected individuals and preventing transmission to others. However, ART does not eliminate all infected cells, allowing the persistence of a small pool of cells collectively known as the HIV reservoir. If ART is interrupted or terminated, the virus will begin replicating again within a couple of weeks because of this reservoir. The reservoir cells are capable of clonally expanding, and surprisingly, not all offspring of a clone exhibit identical levels of viral expression. Developing effective strategies to identify and eliminate such pools of cells is a prevailing challenge in the HIV field. Even the small group of HIV-infected individuals known as “elite controllers” who are able to maintain suppressed viral levels without ART retain a low frequency of intact integrated HIV DNA copies known as proviruses in their peripheral T helper cells.

The Esperanza patient was determined to be an elite controller because she had a very low viral load and no clinical or laboratory signs of HIV-1-associated disease for the entire eight years following her diagnosis, despite receiving no ART during that time. She only underwent ART when she became pregnant, but discontinued treatment after giving birth. To determine whether she had a persistent HIV-1 reservoir, the authors of a recent publication collected blood samples and placental tissue from the patient. They then isolated ~1.2 billion peripheral blood cells and ~0.5 million placental cells from the samples and subjected the cells to amplification and sequencing using primers and probes specific for HIV-1 in a technique that detects single, near full-length HIV-1 proviral genomes. The authors only detected seven proviral HIV-1 DNA species in the blood cells and none in the placenta. However, each of the seven HIV-1 DNA species was defective: one near-full-length sequence contained mutations that were lethal for the virus, and the other six sequences each contained large deletions. Three of these six sequences with deletions were completely identical to each other, suggesting that they were products of clonal expansion. These results distinguished the patient from other elite controllers, indicating that even though she had been infected with HIV-1 at some point and viral replication had occurred in the past, all viral DNA resulting from recent replication cycles was damaged.

The patient’s peripheral blood cells were also used to isolate 150 million T helper cells, which are the primary target of HIV-1. When the authors analyzed these T cells for the presence of replication-competent HIV-1 particles, they did not detect a single virion, a feature that further distinguished the Esperanza patient from other elite controllers, whose blood typically contains up to 50 replication-competent virions per milliliter.

The entry of HIV-1 into cells requires the presence of two cell surface proteins: the receptor CD4, and one of two co-receptors, either CXCR4 or CCR5. Individuals with a CCR5 Δ32/Δ32 genotype, which signifies a mutation in both copies of the gene encoding CCR5, are resistant to HIV-1 infection. Analysis of T helper cells isolated from the Esperanza patient revealed that they fully expressed both wild-type versions of CCR5 and CXCR4 co-receptors, and when tested in vitro, these cells were able to support HIV-1 infection and replication. This observation suggests that the patient was not resistant to infection. However, her serum did not contain the entire antibody profile usually found in HIV-1-positive patients, implying that even though she became infected and replicated virus, she never developed a full HIV-1-specific antibody response. 

The complete elimination of all virus-carrying cells in the context of HIV infection is termed a “sterilizing cure,” and the mechanism responsible for this exceedingly rare phenomenon is unclear. The human immune proteins APOBEC3G and APOBEC3F are known to induce destructive nucleotide changes in the HIV genome, and the authors hypothesize that the lethal mutations found in the near full-length HIV-1 proviral sequence were likely induced by these immune proteins. However, it is unclear why the overall number of proviral species was so low.

Whether or not the Esperanza patient will remain permanently free of HIV is currently unclear. The authors are careful to note that “absence of evidence for intact HIV-1 proviruses in large numbers of cells is not evidence of absence of intact HIV-1 proviruses.” Nevertheless, this study suggests that a sterilizing cure of HIV-1 infection is possible, even if it is rare. The authors hope that additional data collected from the San Francisco and Esperanza patients will provide further insight into the mechanism responsible for a sterilizing cure, which might lead to treatments that cause the immune system to mimic the responses observed in these two patients.

[This article was written in honor of World AIDS Day, which occurs annually on December 1.]

T Cell Responses to Coronavirus Infection are Complicated

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by Gertrud U. Rey

Throughout the current pandemic, there has been a lot of talk about T cells and their role in protecting against SARS-CoV-2 infection and disease. Some data suggest that 20-50% of people with no prior exposure to SARS-CoV-2 have T cells that recognize SARS-CoV-2 peptides, and that these T cells may be a result of recent infections with one or more of the seasonal human coronaviruses. However, it is unclear whether these “cross-reactive” T cells actually protect from SARS-CoV-2 infection and disease.  

T cells are an important part of the adaptive immune response, which initiates during a first exposure to a pathogen and protects from re-infection and disease upon a second exposure to the same pathogen. During that first exposure, T helper cells sense the presence of one or more proteins (i.e., antigens) on the surface of the invading pathogen and release a variety of signals that ultimately stimulate B cells to secrete antibodies to those antigens, white blood cells to destroy ingested microbes, and cytotoxic T cells to directly kill infected target cells (see schematic). Before T cells encounter their first antigen, they are considered to be “naïve.” Upon their first contact with an antigen, they begin to mature and differentiate into either cytotoxic T cells or memory T cells. As we age and encounter more and more pathogens, the ratio of our memory T cells to our naïve T cells increases – a phenomenon sometimes referred to as “immunological age.” 

Based on evidence from several labs, some have suggested that pre-existing cross-reactive memory T cells in people with no prior exposure to SARS-CoV-2 may have a protective effect. However, recent findings indicate that this may not be the case.

Several research groups from the U.S. and Australia analyzed blood samples from individuals with no prior SARS-CoV-2 exposure with the intent of better defining the range of T helper cells that can recognize antigenic portions known as epitopes in the SARS-CoV-2 genome. To ensure that the blood donors had never been infected with SARS-CoV-2, the researchers used stored samples that had been collected between 2015 and 2018. The authors found that the blood samples contained T cells that can recognize SARS-CoV-2 sequences that have at least 67% similarity to seasonal coronavirus sequences. However, the authors also found that people who had experienced a previous infection with SARS-CoV-2 had stronger and more specific (i.e., higher avidity) memory T cell responses to SARS-CoV-2 peptides than people with no prior exposure, and more than half of these responses were directed to epitopes in the spike protein. This suggests that SARS-CoV-2 memory T helper cells preferentially target viral proteins that are made in abundance during infection. 

The authors conclude that an infection with a seasonal coronavirus may induce a range of memory T cells that have substantial cross-reactivity to SARS-CoV-2. However, they are careful to note that the clinical relevance of these data remains unclear and that there is no evidence that these memory T cells have any functional role in protecting from infection or disease.  

Based on these findings, several groups of investigators in Germany wanted to determine whether the observed pre-existing T cell memory response in people with no prior SARS-CoV-2 exposure is protective against COVID-19. They first tested T cell activity in the blood of donors with and without prior exposure to SARS-CoV-2 by exposing their blood to highly immunogenic SARS-CoV-2 peptides. Blood from donors with a prior exposure contained memory T cells that recognized SARS-CoV-2 peptides very well, especially peptides derived from the spike, membrane, and nucleocapsid proteins. Blood from donors with no prior exposure also contained low levels of SARS-CoV-2-reactive memory T cells, but this reaction was more scattered and directed against multiple viral proteins. 

To further characterize the pre-existing cross-reactive T cells in blood samples from people with no prior SARS-CoV-2 exposure, the authors compared the numbers of memory T cells and naïve T cells in the blood samples by analyzing them for the presence of protein markers that are characteristic for each type of cell. A substantial portion of SARS-CoV-2-reactive T cells from donors with no prior exposure were naïve T cells, whereas from COVID-19 patients most were mature memory T cells. Because memory T cells are more easily activated following an infection than naïve T cells, the authors speculate that deficient, low avidity memory T cells in people with no prior SARS-CoV-2 exposure may compete with naïve T cells and prevent their activation and maturation into highly specific memory T cells upon infection with SARS-CoV-2. This could potentially lead to an inferior immune response in people with no prior SARS-CoV-2 exposure. 

Some have suggested that young patients and children may be particularly well protected from SARS-CoV-2 infection and/or disease because they are frequently infected with seasonal coronaviruses and thus presumably have high levels of pre-existing memory T cells. However, the authors found that compared to young people, older people with no prior SARS-CoV-2 exposure actually had higher numbers of SARS-CoV-2 cross-reactive memory T cells, but these T cells had a decreased avidity to SARS-CoV-2 epitopes compared to memory T cells from people with prior SARS-CoV-2 exposure.   

A further comparison of T cell responses in patients with mild or severe COVID-19 revealed that although the latter had high numbers of SARS-CoV-2 specific T cells, these T cells had reduced target specificity and avidity compared to T cells from patients with moderate disease. The authors conclude that this unfocused response may result from recruitment of a broad range of pre-existing memory T cells in people with increased immunological age and may contribute to development of severe COVID-19 in the elderly. 

The initial discovery of SARS-CoV-2-specific memory T cells in individuals with no prior exposure to SARS-CoV-2 had inspired the hypothesis that these T cells could possibly protect these individuals from disease and might partially explain why children are less susceptible to COVID-19. However, in light of these new findings it is likely that pre-existing SARS-CoV-2-specific memory T cells may contribute to the wide spectrum of disease severity among the general population and may actually be partially responsible for severe COVID-19 in the elderly. 

[For an in-depth discussion of these two papers, I recommend TWiV 657 and Christian Drosten’s “Das Coronavirus Update,” episodes 58 and 60.]

How influenza virus infection might lead to gastrointestinal symptoms

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influenza virusHuman influenza viruses replicate almost exclusively in the respiratory tract, yet infected individuals may also have gastrointestinal symptoms such as vomiting and diarrhea. In mice, intestinal injury occurs in the absence of viral replication, and is a consequence of viral depletion of the gut microbiota.

Intranasal inoculation of mice with the PR8 strain of influenza virus leads to injury of both the lung and the intestinal tract, the latter accompanied by mild diarrhea. While influenza virus clearly replicates in the lung of infected mice, no replication was observed in the intestinal tract. Therefore injury of the gut takes place in the absence of viral replication.

Replication of influenza virus in the lung of mice was associated with alteration in the populations of bacteria in the intestine. The numbers of segmented filamentous bacteria (SFB) and Lactobacillus/Lactococcus decreased, while numbers of Enterobacteriaceae increased, including E. coli. Depletion of gut bacteria by antibiotic treatment had no effect on virus-induced lung injury, but protected the intestine from damage. Transferring Enterobacteriaceae from virus-infected mice to uninfected animals lead to intestinal injury, as did inoculating mice intragastrically with E. coli.

To understand why influenza virus infection in the lung can alter the gut microbiota, the authors examined immune cells in the gut. They found that Mice lacking the cytokine IL-17A, which is produced by Th17 helper T cells, did not develop intestinal injury after influenza virus infection. However these animals did develop lung injury.

Th17 cells are a type of helper T cells (others include Th1 and Th2 helper T cells) that are important for microbial defenses at epithelial barriers. They achieve this function in part by producing cytokines, including IL-17A. Th17 cells appear to play a role in intestinal injury caused by influenza virus infection of the lung. The number of Th17 cells in the intestine of mice increased after influenza virus infection, but not in the liver or kidney. In addition, giving mice antibody to IL-17A reduced intestinal injury.

There is a relationship between the intestinal microbiome and Th17 cells. In mice treated with antibiotics, there was no increase in the number of Th17 cells in the intestine following influenza virus infection. When gut bacteria from influenza virus-infected mice were transferred into uninfected animals, IL-17A levels increased. This effect was not observed if recipient animals were treated with antibiotics.

A key question is how influenza virus infection in the lung affects the gut microbiota. The chemokine CCL25, produced by intestinal epithelial cells, attracts lymphocytes from the lung to the gut. Production of CCL25 in the intestine increased in influenza virus infected mice, and treating mice with an antibody to this cytokine reduced intestinal injury and blocked the changes in the gut microbiome.

The helper T lymphocytes that are recruited to the intestine by the CCL25 chemokine produce the chemokine receptor called CCR9. These CCR9 positive Th cells increased in number in the lung and intestine of influenza virus infected mice. When helper T cells from virus infected mice were transferred into uninfected animals, they homed to the lung; after virus infection, they were also found in the intestine.

How do CCR9 positive Th cells from the lung influence the gut microbiota? The culprit appears to be interferon gamma, produced by the lung derived Th cells. In mice lacking interferon gamma, virus infection leads to reduced intestinal injury and normal levels of IL-17A. The lung derived CCR9 positive Th cells are responsible for increased numbers of Th17 cells in the gut through the cytokine IL-15.

These results show that influenza virus infection of the lung leads to production of CCR9 positive Th cells, which migrate to the gut. These cells produce interferon gamma, which alters the gut microbiome. Numbers of Th17 cells in the gut increase, leading to intestinal injury. The altered gut microbiome also stimulates IL-15 production which in turn increases Th17 cell numbers.

It has been proposed that all mucosal surfaces are linked by a common, interconnected mucosal immune system. The results presented in this study are consistent with communication between the lung and gut mucosa. Other examples of a common mucosal immune system include the prevention of asthma in mice by the bacterium Helicobacter pylori in the stomach, and vaginal protection against herpes simplex virus type 2 infection conferred by intransal immunization.

Do these results explain the gastrointestinal symptoms that may accompany influenza in humans? The answer is not clear, because influenza PR8 infection of mice is a highly artificial model of infection. It should be possible to sample human intestinal contents and determine if alterations observed in mice in the gut microbiome, Th17 cells, and interferon gamma production are also observed during influenza infection of the lung.