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Barton Haynes Recently Published Papers and Patent Applications


Haynes recent paper in the August issue of the journal Virology has generated much discussion in the HIV research community, as well as several popular news articles.


Examples of follow-on popular news stories:


HIV Conquers Immune System Faster Than Previously Realised
http://www.docguide.com/news/content.nsf/news/852571020057CCF68525748A00661CD7


HIV Overpowers Immune System Quicker than Previously Thought
http://www.genengnews.com/news/bnitem.aspx?name=38938669


These popular articles do a nice job of summarizing the basic insights found by the Haynes team which shed light on what is responsible for the rapid immune cell death found in early HIV infection. Haynes paper as well as his several patent applications that have recently appeared in the various US and international patent databases provide the all-important details. Haynes work discusses exposed phosphatidylserine (PS) on microparticles overwhelming immune cells, facilitating the rise in viral counts. The following information details this fascinating area of research, and points to this PS-induced immune suppression pathway as being an important mechanism involved in the pathogenesis of a broad range of diseases.



First, some background



Recent insights into enveloped virus infection, from the journal Science

In the April 2008 journal Science, authors Mercer and Helenius discuss data showing how vaccinia viruses (the pox family of viruses), utilize phosphatidylserine (PS) exposed on its surface to facilitate entry into cells. Besides PS aiding viral entry, the authors also discuss recent research showing that exposed PS also prevents an immune response to the virus. They use the term "apoptotic mimicry", (masquerading as a dying native cell), a term showing up more often in recent research involving how various unrelated pathogens use similar means to evade the cells of the immune system, thereby surviving and thriving in the body.


Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host Cells
http://www.ncbi.nlm.nih.gov/pubmed/18436786?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum


short quotes from the paper in Science -

“The induction of blebs, the endocytic event, and infection were all critically dependent on the presence of exposed phosphatidylserine in the viral membrane, which suggests that vaccinia virus uses apoptotic mimicry to enter cells.”

“The Mature Virion membrane has been shown to be enriched in PS and PS is required for infectivity”




The Mercer and Helenius paper spawned several well-written layman's articles on the new insight into viral infection. For instance:


Trojan Horse Of Viruses Revealed
http://www.sciencedaily.com/releases/2008/04/080425065354.htm

excerpts:

ScienceDaily (Apr. 25, 2008) — Viruses use various tricks and disguises to invade cells. Researchers have now discovered yet another strategy used by viruses: the vaccinia virus disguises itself as cell waste, triggers the formation of evaginations in cells and is suspected to enter the cell interior before the immune defense even notices.

The invasion strategy In order to infiltrate a cell, the vaccinia virus exploits the cellular waste disposal mechanism. When a cell dies, other cells in the vicinity ingest the remains, without needing waste disposal experts such as macrophages. The cells recognize the waste via a special molecule, phosphatidylserine, which sits on the inner surface of the double membrane of cells.

This special molecule is pushed out as soon as the cell dies and is broken into parts. The vaccinia virus itself also carries this official waste tag on its surface.

"The substance accumulates on the shell of vaccinia viruses", Jason Mercer explained. The pathogen disguises itself as waste material and tricks cells into digesting it, just as they normally would with the remains of dead cells. As the immune response is simultaneously suppressed, the virus can be ingested as waste without being noticed.




Dirty Rotten Poxviruses
http://sciencenow.sciencemag.org/cgi/content/full/2008/425/1

excerpts:

"poxviruses have engineered a way to sneak into cells through the garbage chute."

"the virus disguises itself as junk"

"the team wondered if the virus was playing dead. They found that the virus's surface was studded with phosphatidylserine, a lipid that also flags dead cells as garbage.

Removing lipids from the virus's surface stopped infection, and recoating the virus with phosphatidylserine
restarted it.

The results suggest that the virus is "more clever than originally thought" because it exploits a garbage-collection process found in almost all cells, says Mercer.





before proceeding further, two words to understand -

Phosphatidylserine: A phospholipid found on the inside of healthy cell walls. As our cells die, they lose the ability to maintain phosphatidylserine (PS) on the inside of the cell wall. PS becomes exposed on our dying cells, and is perceived by cells of the immune system as native / "self" debris, not as a foreign pathogen.

Microparticles: Small vesicles shed from dying cells. Microparticles are made of cell membrane phospholipid molecules. Microparticles expose PS.




A brief primer on phosphatidylserine's role in cell death and immune clearance

As multicellular organisms, we need an immune system that is flexible enough to react differently to foreign invaders as compared to dying native cells. Over the past decade, researchers have shed light on the "fork in the road" which dictates which general type of response our immune system pursues. Phosphatidylserine (PS) is a lipid typically found lining the interior of all healthy cell walls. PS flips to the exterior of the cell membrane as a result of various stresses, and is an early and universal sign of a dying native cell. It is not surprising that exposed PS has recently been shown to provide the fundamental signal for an immune response steered toward homeostasis, a self / maintenance type of immune response. Recent data now illustrates that exposed PS is the impetus that alters the behavior of all the major cell types of the immune system into NOT mounting a specific attack.




Nature finds a way

Scientists have also recently discovered a commonality between cancer, viral, and protozoan parasitic infections. These distinctly different pathogens all exploit exposed PS to aid their survival and proliferation in the body. This common strategy is no surprise when considered with the new understanding of the evolution of metazoan PS mediated immune suppression. Taken together, these recent discoveries suggest that successful pathogens independently evolved to exploit a similar evasion mechanism because it provides the crucial advantage (exposed PS) needed to avoid the immune system's attack.




Where does Barton Haynes fit in?

Over the past couple years, in his work looking into the pathogenesis of HIV, Barton Haynes has been following the hypothesis that the reason HIV manages to proliferate so quickly after infection, and the reason no vaccines have yet been effective, have both been due to a massive amount of "apoptotic debris" (microparticles from dying cells) which occurs early in HIV infection. This debris contains exposed phosphatidylserine (PS), and is responsible for the depletion of CD4+ T cells, as well as an overall blunting of immune response to the virus. Haynes puts forth the idea (documented below) that the fundamental culprit is phosphatidylserine (PS). He cites recent research (Hoffman et al, Interaction between Phosphatidylserine and the Phosphatidylserine Receptor Inhibits Immune Responses In Vivo, The Journal of Immunology, 174: 1393-1404), showing how exposed PS alters the function of macrophages, dendritic cells, and T cells. In the August Journal of Virology he also provides new data showing how microparticles with exposed PS alter the fuctions of antibody producing B cells.





here are earlier excerpts illustrating Haynes/CHAVI overall working hypothesis:


Haynes / CHAVI / Gates hypothesis example 1:

"Their [Haynes group] current hypothesis is that HIV induces a massive apoptosis before and during viral ramp up, and that plasma microparticles (fragments of apoptotic CD3 and T cells) have a suppressive effect on Ab generation, thereby amplifying the apoptotic cascade. "

May 22, 2007
http://www3.niaid.nih.gov/research/topics/HIV/vaccines/advisory/avrs/PDF/AVRS_May07_Summary.pdf




Haynes / CHAVI / Gates hypothesis example 2:

Conclusion: These results demonstrate that at the time of viral load ramp-up in acute HIV-1 infection, there are elevations in plasma levels of TRAIL, FAS Ligand, and TNFR2 that were associated with the presence of microparticles from apoptotic T cells. The presence of these apoptotic markers suggests apoptosis occurring at the time of initial HIV-1 viral load ramp-up. That PS+ apoptotic cells and microparticles have been reported to suppress antigen specific immune responses suggests the hypothesis that immune cell apoptosis in the very earliest stages of acute HIV-1 infection may delay the onset of potentially protective anti-HIV-1 immune responses."

August 20, 2007
http://www.hivvaccineenterprise.org/_dwn/poster_sessions.pdf





Haynes recent publication

The following are excerpts from Bart Haynes' new paper in the August 2008 Journal of Virology, which discuss detailed data showing how exposed PS on microparticles is responsible for the weak immune response to HIV.


Induction of plasma (TRAIL), TNFR-2, Fas ligand, and plasma microparticles after human immunodeficiency virus type 1 (HIV-1) transmission: implications for HIV-1 vaccine design.
http://jvi.asm.org/cgi/content/abstract/82/15/7700


Excerpts:

("MP" = MicroParticles)


“In this study, we raise the hypothesis that in addition to gut CD4 T cell loss, delay in HIV-1 protective immune responses early on after HIV-1 transmission may involve the production of elevated levels of immunosuppressive moieties such as TRAIL, TNFR2 and Fas ligand as well as plasma microparticles.

Microparticles (MPs) are small membrane-bound vesicles that are released from the surface of apoptotic cells by exocytic or budding processes; as such, MPs bear cell surface markers and can bind annexin V because of the expression of phosphatidylserine (32-44, 39).

MPs, which circulate in the blood in many clinical conditions, are part of a spectrum of subcellular structures that are released from cells and can be distinguished from exosomes which are released from multivesicular bodies during activation.

“MPs have immunomodulatory activities and can promote immune cell death; exosomes are also immunologically active, can suppress immune responses (20,34,42,55), and have been reported elevated in chronic HIV-1 (4).”

“suppression of immune responses can be mediated by T cell MPs (32,34,35). CXCR4+ and CCR5+ MPs can transfer co-receptors to coreceptor negative cells, making them susceptible to infection by HIV-1 (48,57).

Phagocytosis of MPs by macrophages releases TGF-beta, prostaglandin E2 and IL-10 that can inhibit antigen specific T and B cell responses (20,35,42). In this regard, Estes et al. have shown dramatic increases in lymph node TGF-beta and IL-10 on day 12 following SIV infection (22).

Importantly, we have demonstrated that PBMC and tonsillar cell MPs can directly inhibit memory B cell activation (Figure 8).”

“it is likely that MPs are responsible for the observed B cell suppressive activity seen in vitro in Figure 8.

In the setting of HIV-1 infection where both activation and apoptosis occur, however, MPs and exosomes may act concomitantly, with exosomes suppressing immune responses (2,7,15,61), and MPs contributing to both immune suppression and cell death (20,32,34,35,39,42,55).”


August 2008, Journal of Virology
http://jvi.asm.org/cgi/content/abstract/82/15/7700






Where Does Haynes Go From Here? Patents Point The Way...

We can see where Haynes is going in his work by looking at two recent patent applications.

In one recent patent application, in the World Intellectual Property Organization database, Haynes discusses what a future successful HIV vaccine must do. He specifically states that any future successful vaccine must stop exposed PS from blunting immune response. He states that an essential goal of a future vaccine is to induce anti-PS antibodies, which would bind/block the PS from downregulating the immune response.

In Haynes second recent patent application, in the US database, he discusses how monoclonal anti-PS antibodies can be safe therapy in people already infected with HIV.




excerpts and links to the two recent patent applications:


The “Vaccine” Patent -

- Thus, HTV virions and HIV envelope can directly induce T cell death in AHI, soluble TRAIL can bind to uninfected cells and induce death in AHI, and with both HTV infection of cells and with massive apoptosis, high levels of phosphatidylserine containing cells and particles likely abound in AHI.

Phosphatidylserine (PS) on the surface of HIV infected cells and virions has been found (Figure 7) and Callahan et al have found PS is a cofactor for HIV infection of monocytes (Callahan, J. Immunol 170:4840 (2003)).

PS-dependent ingestion of apoptotic cells promotes TGF-βl secretion (Huynh et al, J. Clin. Invest. 109:41 (2002)) and interaction between PS and PS receptor inhibits antibody responses in vivo (Hoffman et al, J. Immunol. 174:1393 (2005)).

There are increases in PS+ shed membrane particles in chronic HIV infection (Aupeix et al, J. Clin. Invest. 99:1546 (1997)), and apoptotic microparticles modulate macrophage immune responses (Distler et al, Apoptosis 10:731 (2005)).

The present invention relates to a multicomponent vaccine that addresses problems resulting from the diversity of HIV by the use consensus and/or mosaic HIV genes coupled with strategies designed to break immune tolerance to allow for induction of the desired specificity of neutralzing antibodies at mucosal sites (e.g., through the use of T regulatory cell inhibition and/or TLR-9 agonist adjuvants), and strategies designed to overcome HIV-I induced apoptosis (e.g., induction of anti- phosphatidylserine (PS) antibodies, anti-CD36 antibodies, and/or antitat antibodies).


WHAT IS CLAIMED IS:

1. A method of inducing the production of an immune response against HIV-I in a mammal comprising administering to said mammal: i) a centralized HIV-I gene sequence, ii) an agent that breaks mammalian immune tolerance, and iii) an agent that inhibits HIV-I -induced apoptosis or an immunosuppressive effect of HIV-I -induced apoptosis, wherein (i), (ii) and (iii) are administered in amounts sufficient to effect said production.

22. The method according to claim 1 wherein said agent that inhibits HIV-1-induced apoptosis induces anti phosphatidylserine (PS) antibodies, anti- CD36 antibodies, or anti-HIV tat antibodies.

http://www.wipo.int/pctdb/en/wads.jsp?IA=US2007024122&LANGUAGE=EN&ID=id00000006427540&VOL=87&DOC=0084d3&WO=08/063 586&WEEK=22/2008&TYPE=A2&DOC_TYPE=PAMPH&PAGE=1






The “therapeutic” patent

Here Haynes discusses the use of anti-phospholipid monoclonal antibodies as HIV therapy. Haynes has moved from seeing the lipid binding characteristics of the well-known broadly-neutralizing HIV abs (2F5, 4E10) as unfortunate, and now sees the lipid-binding characteristics as “the key” to broad and safe HIV neutralization. Haynes goes so far as to say that there is no need for an HIV neutralizing antibody to bind a viral epitope. The idea is presented that binding a host-cell 'tag-along' phospholipid results in successful HIV neutralization.


WHAT IS CLAIMED IS:

1. A method of treating HIV comprising administering to a patient in need thereof an antibody derivable from a normal subject or from an autoimmune disease subject that binds to a lipid on the surface of HIV or on the surface of HIV-infected cells and thereby neutralizes HIV-

1, wherein said antibody is administered in an amount sufficient to effect said treatment.

2. The method according to claim 1 wherein said antibody is derivable from an anti-phospholipid syndrome subject.

3. The method according to claim 1 wherein said antibody is non-pathogenic.

4. The method according to claim 1 wherein said antibody is IS1, IS4 or IS6, or binding fragment thereof.

5. The method according to claim 1 wherein said antibody is IS1, or binding fragment thereof.

[0105] ....That IS1 neutralized HIV evidences the facts that: a) humans can make non-pathogenic anti-lipid antibodies that neutralize HIV, and b) IS1 is an antibody that can be safely used as a therapeutic Mab for treatment of HIV infected subjects or in the setting of postexposure prophylaxis of subjects following needle, sexual or other exposure to HIV or HIV infected materials.

http://appft1.uspto.gov/netacgi/nphParser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearchdv.html&r=2&p=1&f=G&l=50&d=PG01&S1=%28%22haynes+barton%22.IN.%29&OS=in/%22haynes+barton%22RS=IN/%22haynes+barton%22





The Big Picture

In the Haynes "therapeutic patent" above, one additional quote stands out:

[0051]" It will be appreciated from a reading of the foregoing that if HIV has evolved to escape the host immune response by making the immune system blind to it, other infectious agents may have evolved similarly. That is, this may represent a general mechanism of escape. That being the case, approaches comparable to those described herein can be expected to be useful in the treatment of such other agents well."


In their recent paper in Science, (the top of this post) Mercer and Helenius also speculate on this broad-based pathogenic immuneevasion mechanism:

Also, in the same issue of Science, Fairn and Grinstein discuss the broader implications of Mercer and Helenius's findings:


A One-Sided Signal
http://www.sciencemag.org/cgi/content/full/320/5875/458

Gregory D. Fairn and Sergio Grinstein


excerpts:

"Because the infected cells undergo apoptosis, and thus experience scrambling of plasma membrane lipids, the budding virus also acquires an envelope that exposes phosphatidylserine on its external surface"

"the presence of exofacial phosphatidylserine is required for viral entry. "

"The involvement of phosphatidylserine may not be limited to infection by vaccinia viruses."

"It is therefore conceivable that HIV similarly requires phosphatidylserine for infection"

"The role of phosphatidylserine in the entry of HIV and other viruses will surely be explored in greater detail now”




ADDENDUM - JULY 24, 2008:

On July 24, 2008, another Haynes patent application went public on the World Intellectual Property Organization database.

Pertinent excerpts follow:


"The time of appearance of antibodies in the development of acute HIV infection has been recently mapped and it has been shown that most of the antibodies arise after a delay in the peak response to HIV envelope epitopes of approximately two to three weeks. Indeed, the most protective antibodies, those that neutralize autologous virus, can be delayed for up to a year."

"To begin to understand the "delay" in induction of antibodies at the time of HIV transmission, the first question to be addressed was whether there are immunosuppressive events, such as massive apoptosis, with release of phosphatidylserine microparticles at the time of viral load ramp up during acute HIV infection."

"Apoptotic microparticles are the products of either activated or apoptotic cells, that are increased in the plasma of a number of diseases, including autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, Crohn's disease, coronary artery disease and other forms of heart disease, and chronic HIV-I infection."

"Apoptotic microparticles can bind to non-apoptotic cells and induce apoptosis, are procoagulant, proinflammatory, and can be immunosuppressive for T and B cell responses to specific antigen."

"Thus, the massive apoptosis that occurs with acute HIV infection with resulting release of TRAIL, mediation of apoptosis via FAS-FASL interactions, and release of PS containing viral and other particles, all conspire to initially immuno suppress the host, preventing rapid protective B cell responses."

http://www.wipo.int/pctdb/en/wads.jsp?IA=US2008000412&LANGUAGE=EN&ID=id00000006622149&VOL=89DOC=00fca1&WO=08/088747&WEEK=30/2008&TYPE=A2&DOC_TYPE=PAMPH&PAGE=1



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