_Going Viral

Jeffrey Jacobson was among the first physicians to take on HIV/AIDS, and over the past 30 years, he had made defeating the disease his life’s work. As his most recent work has shown, he and his colleagues worldwide are getting very close to achieving precisely that.

_Jeffrey Jacobson

Jacobson is a professor of medicine in the Division of Infectious Diseases and HIV Medicine at Drexel University College of Medicine, specializing in infections diseases, internal medicine and HIV.

Jeffrey Jacobson was among the first physicians to take on HIV/AIDS, and over the past 30 years, he had made defeating the disease his life’s work. As his most recent work has shown, he and his colleagues worldwide are getting very close to achieving precisely that.

The first time Jeffrey Jacobson stared into the eyes of an AIDS patient, the disease that was ravaging the victim’s body didn’t yet have a name. The year was 1981, and Jacobson, then a fellow at Mount Sinai Hospital’s School of Medicine in New York City, was studying to be an infectious disease expert.

Maybe it was a fortunate circumstance; a right place, right time type of coincidence. But the emergence of human immunodeficiency virus at a time when he was starting his medical career presented Jacobson and other young physicians like him with a unique opportunity to rewrite the rules for treating patients with chronic infections.

“Suddenly it fell upon us and others to take care of this whole new group of patients,” says Jacobson, who now is chief of the Division of Infectious Diseases and HIV Medicine at Drexel. “Many of us started inpatient and outpatient programs to treat people and study the disease. First we had to figure out what it was. When the virus was discovered, we had to figure out how it was doing its damage and causing all the complicating infections, and develop ways to treat it.”

Despite many medical advances in the treatment of HIV patients, there still is no cure or vaccine for HIV and some 30 years after his career started, Jacobson continues to help patients manage their HIV.

Drexel Medicine’s Partnership Comprehensive Care Practice is the largest HIV patient care center in Philadelphia, but there was little clinical research taking place there when Jacobson arrived six years ago. Since then, Jacobson landed in excess of $10 million in National Institutes of Health funding for a series of studies on improved HIV treatments, novel medicines and potential vaccines. All of the work has but one goal: finally and forever putting an end to the AIDS epidemic.

A Frustrating Start

Pharmacological advances, increased HIV testing and greater public awareness of how HIV disease is spread have helped stabilize the rate of infection and prolonged life expectancy in industrialized countries like the U.S., where resources are plentiful. But HIV remains an almost certain death sentence in many poorer countries and regions of the world, where infection rates and disease burdens are highest. The World Health Organization estimates there were 34 million people living with HIV/AIDS worldwide in 2011, and there were 1.7 million AIDS-related deaths that year.

In the earliest days of the epidemic, Jacobson found that treating his patients also meant confronting issues of HIV stigma and discrimination. He lobbied to increase access to appropriate medical care for infected prisoners. He convinced a PTA group there was no public health danger in allowing an HIV-infected child to attend school. All the while, he and others sought to better understand why the human immune system was not effective at controlling this highly challenging and lethal virus.

“It was very frustrating in the beginning as there were no effective treatments. And because HIV was a viral infection, it wasn’t clear whether there could be very effective treatments anytime soon,” he says.

In tandem with the race to develop safe and effective antiviral agents to kill the virus, infectious disease researchers started looking at different vaccine approaches and ways to manipulate the immune system to make it more active against HIV. The purpose of these early studies was to try to pinpoint immune system deficiencies and determine whether they could be reversed.

“We’ve always been interested in understanding the immunology of HIV disease and seeing if we can improve the immune response,” Jacobson says. “We wanted to know,

“Pills have really penetrated the market. We’ve seen what they can do and they’ve saved lots of lives and they’ve been really phenomenal at turning the epidemic around. But they’re just not enough.”
–Richard Trauger, chief scientific officer at CytoDyn

‘Why was the immune system not effective at controlling this virus, unlike other self-limited viral illnesses that we get?’”

Antiretroviral therapies now in use have been successful at suppressing the virus but do not eradicate the virus from the body. Drugs are designed to attack the virus once it’s invaded healthy cells by affecting enzymes the virus needs to complete its lifecycle. Drug resistance, adverse side effects and toxicities from long-term use remain challenges to managing patients’ care with pills.

Old Science, New Approach

One aspect of Jacobson’s research focuses on an alternative antibody-based approach to treating HIV infection. Antibodies, also called immunoglobulins, are proteins that circulate in the blood stream. They are a natural part of the immune system, helping fight off foreign pathogens that cause disease. When HIV antigens enter the body, the immune system activates white blood cells to create and send out HIV-specific antibodies.

Early antibody-based HIV studies conducted by Jacobson involved collecting hyper-immunoglobulins from the blood of healthy patients and using them to treat patients with more advanced disease. An established practice for more than 100 years, this was an attempt to neutralize the virus. Later, single antibodies with more specific, targeted activity against HIV were constructed. If the antibodies proved effective, it could lead to new vaccine candidates.

This line of research, Jacobson says, evolved into looking for antibodies capable of inhibiting the virus from attaching to and getting inside new cells. HIV antigen can only adhere to a host cell’s outer surface membrane at certain receptor points. By coating these molecules with special antibodies that block HIV—without impairing the receptor’s ability to otherwise function—antibodies essentially work like the lock on the front door, denying HIV the key to gain entry to the cell where it can replicate.

Unlike drugs, which can be swallowed, therapies that use antibodies must be delivered either intravenously or subcutaneously. One of Jacobson’s NIH studies is a Phase II-b clinical trial to optimize the dosing regimen of a novel monoclonal antibody called Pro140. Three initial clinical studies led by Jacobson showed it to be as antiviral as the best oral agents currently in use. Because it’s a molecule produced by the body itself, it’s also less toxic. The most innovative feature of Pro140, Jacobson says, is that it’s longer lasting than drugs currently on the market. One dose of Pro140 was shown to decrease viral loads for at least one to two weeks.

For HIV-infected patients who struggle to take the daily regimen of antiretroviral pills, Pro140 could be a game changer, Jacobson says. The Centers for Disease Control and Prevention estimated that less than 30 percent of the 1.2 million HIV-positive Americans have been diagnosed and treated successfully to the point where their viral loads are undetectable. The main challenge to achieving this clinical result is patient adherence to taking the medication daily as prescribed.

There can be many reasons for non-adherence. In his experience treating HIV patients, Jacobson says, most are young and they struggle to think of themselves as having a chronic disease. Taking a cocktail of pills every day is a reminder that they are different from others. Within the HIV population are subgroups that have particular difficulties, including intravenous drug users and those abused as children.

“It’s hard for anyone to take all their medications all the time but these are people who live disorganized lives to begin with, and that’s how they got infected in first place. They are not necessarily motivated to take care of themselves,” he says.

Stopping the Spread of HIV

Easing the burden of treatment is critical to controlling the spread of HIV because those who engage in high-risk behaviors are at especially high risk of transmitting the virus to others, says Richard Trauger, a former chief scientific officer at CytoDyn, which is hoping to bring Pro140 to market.

“If we can keep people on the therapy, even if they drift off and don’t take their pills, we’ve still got coverage and we’re managing this disease better from an epidemiological perspective,” he says. “Pills have really penetrated the market. We’ve seen what they can do and they’ve saved lots of lives and they’ve been really phenomenal at turning the epidemic around. But they’re just not enough. You can’t give them another pill to solve the [adherence] problem.”

Additional research on Pro140 will explore whether it potentially could be used to block new infections in people for short periods of time, a concept called protective immunity, Trauger says. The earlier clinical studies led by Jacobson show P140 antibodies can remain on cells targeted by HIV for up to 45 days.

During Jacobson’s other NIH Phase II clinical trial, 76 HIV-infected patients who abuse substances and have not been successfully treated with oral drugs will receive a standard drug regimen plus a dose of P140 or a placebo to determine if P140 improves the antiviral response. Researchers will monitor the patients for six months to see if their viral loads reach non-detectable levels.

“Now there’s even more interest in going for a so-called cure,” Jacobson says. “I think the most realistic approach is to go for a functional cure where you don’t fully eradicate the virus, because that’s going to be a tall order.”

Making People Sicker, to Make Them Better

Meanwhile, the search for an effective vaccine continues. Developing a universal HIV vaccine has proven elusive, in part, because there are many different varieties of the virus.

In an immune system-based intervention Jacobson led through the national AIDS Clinical Trial Group, patients whose viral loads had been stabilized with antiretroviral drugs were exposed to their own virus for a short period of time and were asked to stop taking the drugs. This essentially tricked the immune system into fighting a “new” infection. The process was then repeated several times. By monitoring dropping viral loads until they reached a new, hopefully lower set point, researchers were able to gauge for the first time the effectiveness of this and other immune-based approaches.

The results were encouraging. Those who were “pulsed” with their own virus achieved modestly lower viral set points. And about 15-20 percent of the treated group attained near undetectable levels—and stayed there for at least a year. Inducing the immune system to control the virus on its own without the need for antiviral drugs would achieve what has been called a “functional” cure.

“The immune system is not really set up to chronically react to an antigen. It’s set up to see an invader, deal with it and then create memory cells for when it happens again. Otherwise it goes to rest,” Jacobson says. “We thought exposing them again to their own virus for a brief period of time, then treating them to ‘slam down’ the virus would be a way to pulse the immune system without exhausting it too much.”

In another NIH study, Jacobson’s lab is using this new tool of monitoring viral set points to explore the idea of taking samples of a patient’s virus and using it to create a personalized vaccine. First, researchers take blood samples from HIV-infected patients before they are put on antiretroviral therapies. The samples are stored while the patient is treated. Once the virus is suppressed, white blood cells called dendritic cells are harvested from blood samples of HIV-infected patients through a lab process called leukapheresis. In a normal immune response, dendritic cells act to communicate the presence of a foreign antigen to other immune cells which then attack it.

“Dendritic cells are like the guards outside the castle; they signal to everyone else how to respond when there’s an invader,” Jacobson says. “They’re also known as nature’s adjuvant because they boost the response of the immune system to what’s attacking the body.”

By loading the RNA genetic material of the virus onto these active dendritic cells in the lab and transplanting them back into the person’s bloodstream, researchers hope to trigger a stronger, natural immune response, similar to the way vaccines work, but with active disease. This same approach was recently approved to treat prostate cancer, and other research groups are looking at dendritic cell-based vaccines for other types of cancer.

PRO140: Overview

According to the National Institutes of Health, PRO140 is an investigational drug included in the entry inhibitor drug class for the treatment of HIV infection. Entry inhibitors interfere with the first step in the HIV life cycle—binding and fusion to target cells. By preventing HIV from entering target immune cells, entry inhibitors stop HIV from replicating and reduce the amount of HIV in the blood.

‘An Amazing Roller Coaster Ride’

As promising as the research appears, only one HIV-positive adult—the so-called Berlin patient—has been considered functionally cured of the disease; that patient received a bone-marrow transplant for leukemia from a donor genetically resistant to HIV infection, but such treatment is cost-prohibitive and unlikely to be replicated en masse. Meanwhile, in another medical first, doctors announced that a 2-year-old Mississippi child born with HIV and treated aggressively with a full regimen of antiretroviral drugs starting just after birth had also experienced a functional cure. The child had been off drugs for a year with no sign of active virus.

The news was promising, but as Jacobson points it, the baby’s infection may not have been established yet and the drugs may have merely prevented, and not actually treated, the infection.

“Both cases represent situations that are highly unusual and not typical of the average HIV-infected person. Nevertheless, they provide opportunities to advance our knowledge of the mechanisms underlying persistence of HIV infection, knowledge that could help guide the testing of strategies to cure the infection,” Jacobson says.

Each major breakthrough helps rekindle the hope he first felt 30 years ago of helping to find a cure. Great progress has been made in our knowledge and in developing effective treatments for HIV infection.

“It’s been an amazing roller coaster ride. There were a lot of ups and downs and a lot of struggles getting patients the care that they needed, helping them deal with the discrimination and stigma,” he says. “There were a lot of challenges but also many rewards. To have my career span from my training at the beginning of it all to now is amazing.”

_Protecting The Barrier

Thanks to significant strides in HIV research, treatments for the disease have proved to be wildly successful. But as HIV-positive individuals continue to live with the disease, scientists are posed with a new challenge—how HIV affects a now-aging population. Drexel researchers are at the forefront of finding answers and providing solutions.

_Michael Nonnemacher

Nonnemacher is an associate professor in the Department of Microbiology & Immunology.

As the HIV-infected population ages, cognitive issues are arising that affect the quality of life for individuals. To tackle this problem, Michael Nonnemacher, an assistant professor in microbiology and immunology at Drexel University, is developing in vitro models to study the mechanisms of the blood-brain barrier and the impact of HIV on the barrier’s function.

The project also will look at the role aging plays on the barrier’s ability to regulate what gets in and out of the central nervous system through the lens of the viral protein Tat, which is secreted from HIV-infected cells. Tat, research has shown, has a role in weakening the blood-brain barrier and causing inflammation in the central nervous system.

“We’re looking at it specifically from the genetic variation of a protein that has been shown to impact the blood-brain barrier,” Nonnemacher says.

That barrier separates the circulating blood from the brain fluid in the central nervous system and is made up of three compartments: blood, a layer of endothelial cells and astrocyte and pericyte cells in the brain that support the endothelium and effectively tightly stitch together a protective wall.

When working properly, only certain molecules can pass through, such as immune cells that patrol for pathogens, drugs and other small molecules, Nonnemacher says. But in HIV-infected people, the barrier’s permeability is disrupted, likely due to cellular changes.

Cells infected with HIV have altered cytokines, which are proteins that help regulate the immune system. They also produce virus particles and secrete Tat.

Tat, in particular, degrades the tight junction proteins, essentially the astrocytes that hold together the endothelial blood-brain barrier, and disrupts the barrier’s structure. Once it is damaged, cell migration across the wall increases the quantity of infected cells, viral proteins and viruses that reach the central nervous system.

“As these cells senesce, what we don’t know is what kind of loss of function does that provide to the barrier,” says Nonnemacher. “Is there a breach? Or is the barrier intact?”

The genetic variation of Tat, related to the HIV virus’s hallmark ability to mutate, is an important factor in the severity of HIV-related cognitive deficits. Nonnemacher’s project, funded through a $20,000 grant from Temple University’s Comprehensive NeuroAIDS Center, will focus on developing cell models to understand how HIV affects the blood-brain barrier and ultimately the central nervous system, particularly in an aging population.

“We’re asking basic mechanistic questions: Is the barrier that regulates the in and out of the brain differently working in an aged versus non-aged person in an HIV-infected model?”

“We’re asking basic mechanistic questions: Is the barrier that regulates the in and out of the brain differently working in an aged versus non-aged person in an HIV-infected model?” he says.

Patient data suggest the answer is yes, Nonnemacher says. Once researchers figure out what exactly is going on, then the next question that he hopes to attack is this: “Is there something we can do to fix it?”

_Double Whammy

About 30 percent of people worldwide—more than 10 million individuals—are not only infected with AIDS-causing HIV but also with the hepatitis C virus (HCV). That dual whammy takes its toll on the immune system and may accelerate the aging process in those patients, according to Vanessa Pirrone, a research instructor in the Department of Microbiology and Immunology.

_Vanessa Pirrone

Pirrone is a research instructor in the Department of Microbiology and immunology.

About 30 percent of people worldwide—more than 10 million individuals—are not only infected with AIDS-causing HIV but also with the hepatitis C virus (HCV). That dual whammy takes its toll on the immune system and may accelerate the aging process in those patients, according to Vanessa Pirrone, a research instructor in the Department of Microbiology and Immunology.

In a new study supported with University developmen- tal funding, she looks at the impact of co-infection on an aging population. The two viruses “can exacerbate one another,” Pirrone says. Co-infected patients have more rapid progression of liver disease, as well as higher risks for cardiovascular problems, diabetes and carcinomas.

“In general, you have further waning of the immune sys- tem and more immunosenescence,” she says. Normally, the immune system in healthy people slows as they reach their 70s, 80s and 90s. But in the HIV-infected population, problems start to arise when they are only in their 50s. Older HIV-infected patients also suffer two to three times the fre- quency of dementia than younger people with the disease.

Preliminary results from analysis of immune cell and plasma samples from two groups—age 35 and younger and age 50 and older—suggest that co-infection with HCV significantly further accelerates the effects of the aging process in patients with HIV, Pirrone says. She suspects that the two viruses act synergistically by speeding up immune system activation, the excessive and aberrant response of the immune system to HIV that plays a major role in AIDS’ progression.

“The co-infected are not quite as healthy as the mono-infected patients,” she says. “We’re looking to see why that is.” Pirrone’s project will investigate the effect of aging on immune-regulating molecules (cytokines and chemo- kines) as well as on genes, proteins and pathways in those who are co-infected compared to those who are only infected with HIV or HCV.

She will also study different cell populations and determine the mechanisms involved in chronic immune activation and viral immunity in the two age groups.

“It is really untested waters,” Pirrone says.

The study is tapping HIV and co-infected patients who are part of the large group that researchers are monitoring through Drexel’s HIV/AIDS clinic.

Samples of immune cells and plasma from these patients show that those with only HIV infection show increased viral loads as they age, even when they diligently pursue highly active antiretroviral therapy, or HAART. “You would expect the viral load to go down,” Pirrone says, noting that HAARTs have improved lifespans so that more AIDS pa- tients are living into their 50s and beyond. “But the viral load is increasing regardless of the therapy.”

_Rats & ‘Senior Moments’

Surprisingly, few animal models exist to study HIV infection or antiretroviral therapies on the aging brain. Barry Waterhouse, a professor in neurobiology and anatomy at Drexel, hopes to address that pressing concern through the development of a robust rat model.

_Barry Waterhouse

Waterhouse is a professor of neurobiology and anatomy and vice dean of biomedical graduate and postgraduate studies

Researchers think the mix of drugs used to treat HIV/AIDS or the progression of the disease itself is causing inflammation and neurotoxicity in the brain. While anyone growing older suffers cognitive lapses—those so-called senior moments—HIV/AIDS patients appear to experience exacerbated problems.

“While the disease is under control, they’re starting to experience these cognitive deficits,” Waterhouse says. “This is very debilitating, because they’re otherwise healthy and in the workforce but their decision-making processes are compromised.”

Many excellent tissue culture models exist to study these processes, but in vivo options are limited. Waterhouse’s two-year pilot project, funded with a starter grant of $275,000 from the National Institutes of Health, will adapt a rat model that his lab has used in its study of attention deficit hyperactivity disorder to instead look at cognitive skills in relation to HIV/AIDS and aging.

In this project, the rats, trained to do two tasks that involve the prefrontal cortex, are exposed to viral coat proteins, which envelope the virus itself. “The viral coat proteins are not themselves infectious,” Waterhouse says, “but when the virus invades the central nervous system, the proteins become distributed around the brain, and they can generate an immune response from the brain’s tissue. That, it’s believed, precipitates a series of events that leads to cognitive decline and neurotoxicity.”

Two types of attention, sustained and flexible, are being measured.

To evaluate sustained attention, the rats are trained for 2.5 months in a behavioral chamber to recognize whether a dim light that appears for a mere 15 seconds is on or off. If it is on, the rat presses one lever and gets a water reward. If it is off, it presses another lever for its reward.

To measure flexible attention, the rats are trained on a set of rules to retrieve a food reward from a small clay flowerpot. Initially, the animals are trained to key into odor. Once mastered, the odor linked to the reward is changed. Once this new rule is mastered, the reward link is again changed, this time to the digging media in the pot or the textured surface surrounding the pot.

The viral coat protein—gp120 was selected as the most likely culprit—is introduced via surgery into the ventricular system of the brain.

Animals, both adult and aged, will be tested on the cortex dependent behavioral tasks both before and after the infusion of gp120.

Very preliminary results show that adult animals with gp120 have flexible attention deficits, Waterhouse says. “They’re slower to change their behavior patterns,” he says.

_Cognitive Secrets

The retrovirus that causes AIDS acts like a shape-shifting villain. As HIV replicates over time, random mutations take place in the viral genome—one of the hallmarks of the disease that makes containing and curing it such a challenge.

_Brian Wigdahl

Wigdahl is a professor and chair of the Department of Microbiology and Immunology.

The retrovirus that causes AIDS acts like a shape-shifting villain. As HIV replicates over time, random mutations take place in the viral genome—one of the hallmarks of the disease that makes containing and curing it such a challenge.

“We’ve been very interested in studying viral evolution and the structure of the virus, from the beginning, most earliest stages all the way through the end, when someone dies from immune system dysfunction, the virus in the brain, with neurologic problems,” says Brian Wigdahl, chair of the Department of Microbiology and Immunology and the director of the Institute of Molecular Medicine and Infectious Disease.

In a $3.5 million NIH-supported research project, Wigdahl focuses on what happens to the replication process as patients live longer thanks to combination drug therapy. His lab also is interested in how the virus affects the aging process.

To study these issues, blood samples from a large group of patients—more than 500 people—seen at Drexel’s HIV/AIDS clinic will be analyzed over time. (It is the largest clinical population being monitored long-term in the Philadelphia area, Wigdahl says.)

“We’ve taken HIV disease and with antiretroviral drug therapy, we’ve converted it into a chronic condition.”

In this project, his hypothesis is that viral and host genetic factors account for aging AIDS patients’ susceptibility to neurocognitive problems. “My interest is starting to really characterize the molecular architecture of the viral genome,” he says. “It’s a study of detail.”

But the payoff is potentially huge, Wigdahl says. It could lead to therapies targeted at new viral genes and proteins, including those related to cognitive decline. Even though about 30 drug treatment protocols for AIDS already exist, “it is still important to keep developing new therapies because of drug resistance and toxicity,” he says.

Wigdahl, who has studied HIV/AIDS since the mid-1980s, also still holds out hope for finding a cure. “That’s a major point of emphasis with a number of groups, including our own,” he says.

In the U.S., more than 1.1 million people are living with HIV infection, according to the Centers for Disease Control. Researchers estimate that by 2015, half of the infected population will be older than age 50.

“It’s a good problem to have,” Wigdahl says. “We’ve taken HIV disease and with antiretroviral drug therapy, we’ve converted it into a chronic condition.”

In response, his research is shifting gears from a younger population of study to an older one, with a focus on how AIDS will progress in that aging environment, where normal declines in the immune system (immunosenescence) and cognitive performance are already underway.

One area of investigation is viral single nucleotide polymorphism, or the genetic variation in a DNA sequence. A person with HIV infection has thousands of viral cells, each with a slightly different genetic makeup, the so-called viral swarm. But interestingly, during the early hours up to a few weeks after initial transmission from one individual to another, “a serious bottle neck occurs,” Wigdahl says. “What really gets transmitted to that individual is a single genotype.”

_Intricate Transmission

Despite 30 years of study, male-to-female transmission of the virus that causes AIDS is not fully understood. Even less is known about infection risk within an aging population.

_Fred Krebs

Krebs is an associate professor in the Department of Microbiology and Immunology Department.

Despite 30 years of study, male-to-female transmission of the virus that causes AIDS is not fully understood. Even less is known about infection risk within an aging population.

Given the global AIDS epidemic among heterosexuals as well as longer lifespans for those with AIDS, a better understanding of HIV transmission and the impact of aging on the disease progression is crucial, says Fred Krebs, an associate professor in microbiology and immunology at Drexel. He has proposed exploring the effect of age on factors in seminal fluid that modulate the female reproductive tract’s immune response. That in turn could alter the risk of HIV infection.

“It’s an area that has not been studied much at all, in any population, at any age,” he says.

Until recently, it was thought that the static medium of semen deposited the virus and infected cells into the vaginal environment, from where the disease spread systemically. In fact, semen contains numerous active factors, such as cytokines and chemokines, that prepare women for reproduction by changing the immune response in the female reproductive tract to allow for the foreign antigen, semen.

“The immune system is dialed back to create an environment of tolerance,” Krebs says. While good for conception, this process could increase the risk of HIV infection, he says.

In addition, mouse models have shown that the introduction of seminal fluid into the female reproductive tract results in inflammation, which causes the recruitment of immune cells to the area.

“The immune response in the female reproductive tract is really a double-edged sword,” Krebs says. “It could increase risk of transmission. It also could decrease risk of transmission.” How? More immune cells could fight off an HIV infection, or they could heighten risk by offering more targets for infection.

Aging, it is suspected, further impacts transmission scenarios. It’s an important aspect to explore for a number of reasons, Krebs says.

AIDS is no longer a death sentence within 10 years of infection because of effective combination antiretroviral therapies. American society encourages and values sexuality among older people—increasing the risk of transmission among this aged population. Finally, immunosenescence, a natural process in which the immune system deteriorates over time, likely affects the transmission risk of HIV.

In the case of immunosenescence, the body becomes “less efficient in fighting off disease and pathogens and less efficient at controlling infection that has already taken root,” Krebs says.