HIV vaccine production

Research on HIV over the past decade has led to many promising ideas for vaccines to prevent infection by the AIDS virus, but very few candidate vaccines have been tested in clinical trials. One reason for this is the technical difficulty of manufacturing vaccines based on the envelope proteins of the virus, according to Phil Berman, who led development of a major component of the only vaccine to have shown any efficacy against HIV in a clinical trial.

Berman, the Baskin Professor of Biomolecular Engineering at UC Santa Cruz, has now developed new methods for the production of HIV vaccines. His approach solves major technical problems which have bedeviled the field. Berman described the new methods, and the candidate vaccines his lab has produced.

Researcher was able to use robotics to shorten the time required to produce stable cell lines, needed to make the proteins for a vaccine, while at the same time greatly increasing how much of the protein the cell lines can produce. The improved yield makes it possible to reduce the size of the bioreactor needed to make vaccine for large clinical trials—from 200- to 10,000-liter vessels to 50- or 100-liter vessels—resulting in tremendous savings in the equipment required and cost of materials.

They were  able to create cell lines that make HIV envelope proteins with the right kind of carbohydrate components (called glycans) needed for an effective immune response. The carbohydrates attached to the protein are really important, something no one realized until recently. The conventional way of making these envelope protein vaccines incorporated the wrong kind of carbohydrates.

Cell lines derived from Chinese hamster ovary (CHO) cells are the industry standard used to produce complex recombinant proteins for therapeutic use. These cells are also useful for the production of HIV vaccines. The gene for the desired protein is transferred into CHO cells in a process called transfection, and tens of thousands of transfected cells are screened to find the few rare cells that produce large amounts of the protein. The best cell lines are grown in large batches, in a process similar to yeast fermentation for making beer, and the proteins are then isolated and purified.

Berman's lab developed a new robotic method to isolate high-producing cell lines making HIV envelope proteins. This shortened the time required to produce stable cell lines from 18 to 24 months to just 2 or 3 months, while increasing yields by a factor of 100 to 200. Those improvements are in comparison with Berman's previous experience creating the AIDSVAX vaccine, first at Genentech and then at VaxGen.

Fluorescent antibodies were used to label the HIV gp120 protein and identify high-producing cell lines. Credit: Phil Berman, UCSC. AIDSVAX was one component of an experimental vaccine regimen used in a large-scale clinical trial known as RV144, which showed 31 percent efficacy in preventing new HIV infections. The RV144 results showed that protection was correlated with antibodies to a certain segment of an HIV envelope protein called gp120.

 The original AIDSVAX vaccine had the completely wrong type of carbohydrate, and that we might improve the level of protection if we could find a way to make it with the proper type of carbohydrate. They set out to create a cell line that can produce the unusual glycans found on HIV envelope proteins rather than the complex glycans CHO cells normally produce. This was made possible by the powerful new gene editing technology known as CRISPR/Cas9.

 They used CRISPR to create a new cell line they called MGAT CHO, which produces proteins lacking complex glycans containing sialic acid and enriched for the simple "high mannose" type found on HIV envelope proteins. An unexpected benefit of this new cell line was that it enabled a simpler, less expensive process for recovering and purifying the proteins.

Carbohydrates may not be immunogenic, but HIV turns out that the most important antibodies are directed to carbohydrate. It leads to an improved version of the vaccine used in the RV144 trial. Berman's lab currently has two cell lines he said are ready to start producing vaccines on a large scale. He is now looking for partners and funding to bring them into clinical trials. Researchers have continued to use the AIDSVAX vaccine in clinical studies because it has been so hard to make new HIV vaccines.
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Latent HIV reservoirs show resistance to white blood cells

A recent study by researchers at the George Washington University (GW) found that latent HIV reservoirs show resistance to CD8+ T-cells, a type of white blood cell whose primary function is to kill infected cells.

According to Brad Jones, PhD, primary author of the study and assistant professor of microbiology, immunology, and tropical medicine at the GW School of Medicine and Health Sciences, researchers have identified a barrier.

It is difficult to understand the nature of that barrier, they used the most powerful combinations against these cells, and when the dust settled they found that the virus was present at just as high levels as what was started with.

HIV/AIDS treatment currently includes lifelong, antiretroviral therapy while the search for a cure continues. Persistent, latent reservoirs of the virus make efforts to cure infection difficult. In order to eradicate those HIV reservoirs, researchers must find a way to eliminate persistent populations of cells with integrated HIV proviruses.

The paradigm is aimed at combining latency reversing agents (LRA) with immune effectors, such as T-cells, to wake up the virus and kill the reactivated cells.The study found that latent HIV reservoirs exhibit inherent resistance to CD8+ T-cells.

The team conducted their research using the CD8+ T-cells of people living with HIV, in the combination with LRAs to attack and kill the infected cells. The results suggest that cells infected by replication-competent HIV possess inherent resistance to the T-cells, which present an obstacle on the road to curing HIV.
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Gene therapy can destroy HIV infected cells

Through gene therapy, researchers engineered blood-forming stem cells (hematopoietic stem/progenitor cells, or HSPCs) to carry chimeric antigen receptor (CAR) genes to make cells that can detect and destroy HIV-infected cells. These engineered cells not only destroyed the infected cells, they persisted for more than two years, suggesting the potential to create long-term immunity from the virus that causes AIDS.

Antiviral drugs can suppress the amount of HIV in the body to nearly undetectable levels, but only an effective immune response can eradicate the virus. Researchers have been seeking a way to improve the body's ability to combat the virus by engineering blood-forming stem cells to specifically target and kill HIV-infected cells for the life of the individual.

 Although chimeric antigen receptor (CAR) T-cells have emerged as a powerful immunotherapy for various forms of cancer -- and show promise in treating HIV-1 infection -- the therapy may not impart long-lasting immunity. Researchers, physicians and patients need T cell-based products that can respond to malignant or infected cells that may reappear months or years after treatment.

Because HIV uses CD4 to infect cells, the researchers used a CAR molecule that hijacks the essential interaction between HIV and the cell surface molecule CD4 to make stem cell-derived T-cells target infected cells. When the CD4 on the CAR molecule binds to HIV, other regions of the CAR molecule signal the cell to become activated and kill the HIV infected cell.

The researchers found that, in test animals, modification of the blood-forming stem cells resulted in more than two years of stable production of CAR-expressing cells without any adverse effects. In addition, these cells were widely distributed throughout the lymphoid tissues and gastrointestinal tract, which are major anatomic sites for HIV replication and persistence in infected people. Most important, engineered CAR T-cells showed efficacy in attacking and killing HIV-infected cells.
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Treat sepsis with vitamins C

Sepsis is a progressive disease process caused by an aggressive and dysfunctional immune response to an infection in the bloodstream. Illnesses like bronchitis, pneumonia and kidney infection can turn septic.

 Sepsis starts with symptoms of an infection, the condition can progress to septic shock, which may be lethal.
Lack of vital vitamins in the body can increase the risk of Sepsis.

Chronic diseases like AIDS, cancer, diabetes, lung, kidney and liver raises the risk of Sepsis. Being exposed to
infection-causing bacteria in health centers can also increase the risk of Sepsis.

The condition can leads to extremely low blood pressure that is unresponsive to fluid replacement, weakening of the heart and multiple-organ failure if treatment is delayed.

Steroid hydrocortisone, intravenous   (IV) vitamin C, thiamine (vitamin B-1) can be used to treat Sepsis. Vitamin C
prevents and treat infectious diseases.
Thiamine is required for metabolism of some of the metabolites of vitamin C. It
reduces the risk of renal failure and mortality.
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Patentiflorin A more effective for HIV treatment than AZT

Patentiflorin A, is a chemical derived from the willow-leaved Justicia, It is a compound in a plant found in Southeast Asia.

AZT is an anti-viral drug that reduces the amount of HIV virus in the body and reduces the risk of developing AIDS.

Pateniflotin A is more effective than
AZT for treating HIV virus at the early stage of the infection and when the
virus enters macrophage cells.

It's also effective against drug resistance strains of HIV virus, it may be useful in future for producing better HIV drug.

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Antiretroviral therapy increases life span of HIV patients

Life expectancy for people with HIV has increased by 10 years since the introduction of antiretroviral therapy.

Antiretroviral therapy has been used to treat HIV for 20 years, but newer drugs have fewer side effects, involve taking fewer pills, prevent multiplication of the virus.

Regular treatment, increased use of screening and prevention programmes for conditions such as cardiovascular disease and cancer have also contributed.

Antiretroviral therapy first became widely used in 1996. It involves a combination of three or more drugs that block the HIV virus from increasing.

It also prevents onward spread of the disease. The World Health Organization  (WHO) now recommends antiretroviral therapy to be given as soon as possible after diagnosis to all people with HIV.

When looking specifically at deaths due to AIDS, the number of deaths during treatment reduced over time between 1996 and 2010, likely as a result of newer drugs being more effective in restoring the immune system.

As a result of the improvements, between 1996 and 2013, the life expectancy of 20-year-olds treated for HIV increased by nine years for women and 10 years for men.

HIV levels and immune activation not better during antiretroviral treatment

Rajesh Gandhi of Massachusetts General Hospital, Boston and colleagues in the AIDS Clinical Trial Group ACTG  tested HIV patients to know if elevated immune activation is driven by the low levels of HIV is present in patients undergoing treatment.

They used blood samples of people who took part in ACTG clinical trials, measured molecular markers of HIV, immune system activation and inflammation in 101 people before and during antiretroviral drugs treatment for 7 years.

Researchers discovered connection between HIV levels, immune activation and inflammation in HIV patients

The link does not continue during treatment, this shows that elevated immune activation during treatment is not driven by HIV in the blood.

People with higher HIV levels before treatment may have higher HIV levels during treatment.