Following Article by Jon Cohen

Three back-to-back talks at a meeting earlier this month provided fresh insights into why HIV is so difficult to eliminate from the body: Even when antiretroviral drugs (ARVs) knock down HIV to undetectable levels in blood, the virus remains active in tissues. Sparked in part by the apparent cure of the “Berlin patient,” many of those attending the meeting are exploring ways to seek out and destroy the tiny amounts of HIV left in the bodies of people who take powerful ARVs. The main obstacle to a cure is that reservoirs of cells that harbor latent HIV still persist.

HCV replicates extremely rapidly. Theoretically, one live HCV virus can replicate itself into one trillion viral particles in a day. It would be nearly impossible to kill all HCV in one’s body with the existing FDA-approved antivirals, including the new protease inhibitors. We need to also rely on our immune system to contain and eventually eradicate the HCV invaders from our body, including the undiscovered hiden areas in the boldy, like lymphatic nodes.

I plans to collaborate with the manufacturers of protease inhibitors to incorporate HepC Herba™ and P-10 Herba™ with their protease inhibitors in combination with the interferon and ribavirin combination treatments to help people with HCV infections, including those who have chronic hepatitis C and HCV carriers. The use of HepC Herba™ and P-10 Herba™ shall help enhance the containment of the viruses at potential undetectable levels through long-term maintenance usages once the virus has been controlled mostly and quickly through the protease inhibitors. HepC Herba™ and P-10 Herba™ can also protect the liver, restore and maintain normal liver function possibly throughout the natural life expectancy, and avert most of the severe adverse side effects from the protease inhibitors and interferon and ribavirin combination treatments.

Our Taiji for Chronic Pain Management Workshop program using taiji chuan and neigong relaxation exercise and meditation may have a very good chance to help on the depression and suicidal tendance adverse side effects from the conventional antiviral drug treatments. We now may have a good chance toward a cure or at least quasi cure for the chronic hepatitis C patients and the HCV carriers. I will raise funds to do the work towards this goal.

You are welcome to spread the news and my e-mail to all your contacts who have HCV infection, either with chronic hepatitis C or being the HCV carriers. They are welcome to send in their inquiries and questions to me directly through the e-mails or letters or through you. Good luck!

Sincerely, Shie-Ming Hwang, Ph.D.

Reference:  http://ingoodhealth.blogspot.com/

The pre-formulations used for the development of HepC Herba™ had been tested against a murine retrovirus and were found to be active against Ecotropic Murine Leukemia Virus (EMLV) in vitro before they were tested active against HIV-1 exo vivo.

Please see the U.S. Patent 5,989,556, Example 4, Table 2 on page 23.

HepC Herba™ has also been found to help those who have chronic fatigue from chronic hepatitis C and mononucleosis from Epstein-Barr virus infection. Those who took HepC Herba™ generally have more energy after two weeks of taking the formulation three times a day orally like drinking a coffee.

Sincerely,

Shie-Ming Hwang, Ph.D.

Good!

Please contact Dr. Judy A. Mikovits, the senior author of the article, at the Whittemore Peterson Institute, Reno, NV, through her e-mail at judym@wpinstitute.org. I would suggest her to look into the sensitivity of the XMRV (xenotropic murine leukemia virus-related virus) Assay among the laboratories who showed contradictory results for the detection of XMRV in CFS (Chronic Fatigue Syndrome) patients’ samples.

To confirm her discovery of the linkage between XMRV and CFS, she needs to demonstrate the decrease or eradication of XMRV in CFS patients before and after recovery from their CFS. HepC Herba’s herbal ingredients in their other combinations with other herbal ingredients had been shown to be active against Ecotropic Murine Leukemia Virus (EMLV). HepC Herba™ itself had also been shown to help those with chronic hepatitis C along with chronic fatigue to feel with more energy in two to three weeks of taking the product.

I suggest Dr. Mikovits or some scientist(s) to conduct a clinical research using HepC Herba™ to treat people with chronic fatigue syndrome (CFS) or chronic fatigue and immune dysfunction syndrome (CFIDS) for one to two months, and follow the assay of their peripheral blood mononuclear cells (PBMCs) for XMRV levels in conjunction with their recovery or partial recovery from CFS or CFIDS in two to four weeks (past experience). A parallel control study with healthy people should also be conducted for comparison.

This should settle the contradictory findings. This study may also help those with prostate cancer or prevent prostate cancer from XMRV, since it had been shown that XMRV could infect human cells and had previously been linked to prostate cancer.

I am planning to organize a research team to respond to the NIH’s Martin Delaney Collaboratory: Towards an HIV-1 Cure (U19). The Letter of Intent is due October 4, 2010. The Application is due November 4, 2010. The Funds available is $8.5M for fiscal year 2011 for 1-2 awards. Direct cost may range from $3 to 5 million per year for up to five years, i.e., $15 to 25 millions total for five years. More funds may be available when needed and can be justified.

The Collaborative Agreement requires two research projects and one administrative core. A research core is optional if needed. An education and research institute and a private sector are required in the collaborative agreement. One of them shall function as the primary applicant. Sage R&D can function as the primary applicant and collaborate with a university or a medical center with HIV/AIDS research program.

One research project I am considering is a basic research to study the immune response of HepC Herba™ or P-10 Herba™, and their combination. Another research project shall be a clinical research on using HepC Herba™, with or without P-10 Herba™, for treating HIV/AIDS patients with conventional antiretroviral combination therapy to see if the use of HepC Herba™ or in combination with P-10 Herba™ would: (1) alleviate the severe adverse side effects of the combination antiretroviral therapy, (2) protect the liver from the damaging effect of the antiretroviral drugs for long-term use, (3) sustain the undetectable viral load on the long-term basis to replace the much less tolerable, much less compliable, and much more costly antiretroviral combination therapy, and (4) prevent or treat HIV-AIDS related complications. This shall constitute a partial cure, if not the complete cure, to the HIV-1 problem.

I am considering to have you get involved in the core administration on the “Towards an HIV-1 Cure Collaborative Agreement with NIH” to assist me to pull the research team together and manage the supply of the HepC Herb™ and P-10 Herba™ to the researchers as needed. You are also authorized to contact Dr. Judy A. Mikovits and other researchers and venture capitalists or foundations on the potential collaboration and funding or venture for CFS or CFIDS and Prostate Cancer prevention and treatment. Please let me know. Thanks for your continuing support.

Sincerely,

Shie-Ming Hwang, Ph.D.

Thanks for the information. We already know the linkage between chronic hepatitis B and chronic hepatitis C and the liver cancer. HepC Herba™ has been shown to help restore the liver function, reverse liver cirrhosis possibly through liver regeneration by itself while modulate the chronic inflammation of the liver and stop the progression of the chronic liver inflammation disease. I expect HepC Herba™ would help prevent liver cancer by calming down the chronic liver inflammation which can lead to deadly liver cirrhosis and liver cancer.

However, the presence of the hepatitis B virus or hepatitis C virus alone in the liver, for those who are the carriers, can still lead to liver cancer. HepC Herba™ has been shown to reduce the viral load in people with chronic hepatitis C. We still do not know how low the viral load should be so the presence of the virus would not lead to liver cancer. A retroactive research is needed to review the viral load of chronic hepatitis C patients who had developed liver cancer as compared to those who had not. We need to find or encourage a scientist to apply for a NIH grant to do this.

For those carriers, they still need to be monitored periodically, say every 4 to 6 months, for potential development of chronic hepatitis or liver cancer. This can be done throught routine blood test for liver enzymes panel, including AST (SGOT), ALT (SGPT), alkaline phosphatase, and alpha-feto protein.

You may want to put my comment in your website in FAQ section.

Sincerely,

Shie-Ming Hwang, Ph.D.

Sequences of full-length XMRV genomes from two CFS patients and a partial genome from a third patient were generated (table S1). CFS XMRV strains 1106 and 1178 each differed by 6 nt from the reference prostate cancer strain XMRV VP62 (EF185282 [GenBank] ), and with the exception of 1 nt, the variant nucleotides mapped to different locations within the XMRV genome, suggesting independent infections. In comparison, prostate cancer–derived XMRV strains VP35 and VP42 differed from VP62 by 13 and 10 nt, respectively. Thus, the complete XMRV genomes in these CFS patients were >99% identical in sequence to those detected in patients with prostate cancer. To exclude the possibility that we were detecting a murine leukemia virus (MLV) laboratory contaminant, we determined the phylogenetic relationship among endogenous (non-ecotropic) MLV sequences, XMRV sequences, and sequences from CFS patients 1104, 1106, and 1178 (fig. S2). XMRV sequences from the CFS patients clustered with the XMRV sequences from prostate cancer cases and formed a branch distinct from non-ecotropic MLVs common in inbred mouse strains. Thus, the virus detected in the CFS patients’ blood samples is unlikely to be a contaminant.

To determine whether XMRV proteins were expressed in PBMCs from CFS patients, we developed intracellular flow cytometry (IFC) and Western blot assays, using antibodies (Abs) with novel viralspecificities. These antibodies included, among others, (i) rat monoclonal antibody (mAb) to the spleen focus-forming virus (SFFV) envelope (Env), which reacts with all polytropic and xenotropic MLVs (7); (ii) goat antisera to whole mouse NZB xenotropic MLV; and (iii) a rat mAb to MLV p30 Gag (8). All of these Abs detected the human VP62 XMRV strain grown in human Raji, LNCaP, and Sup-T1 cells (fig. S3) (5). IFC of activated lymphocytes (6, 9) revealed that 19 of 30 PBMC samples from CFS patients reacted with the mAb to MLV p30 Gag (Fig. 2A). The majority of the 19 positive samples also reacted with antisera to other purified MLV proteins (fig. S4A). In contrast, 16 healthy control PBMC cultures tested negative (Fig. 2A and fig. S4A). These results were confirmed by Western blots (Fig. 2, B and C) (6) using Abs to SFFV Env, mouse xenotropic MLV, and MLV p30 Gag. Samples from five healthy donors exhibited no expression of XMRV proteins (Fig. 2C). The frequencies of CFS cases versus healthy controls that were positive and negative for XMRV sequences were used to calculate a Pearson ² value of 154 (two-tailed P value of 8.1 x 10^–35). These data yield an odds ratio of 54.1 (a 95% confidence interval of 23.8 to 122), suggesting a nonrandom association with XMRV and CFS patients.

To determine which types of lymphocytes in blood express XMRV, we isolated B and T cells from one patient’s PBMCs (6). Using mAb to MLV p30 Gag and IFC, we found that both activated T and B cells were infected with XMRV (Fig. 2D and fig. S4A). Furthermore, using mAb to SFFV Env, we found that >95% of the cells in a B cell line developed from another patient were positive for XMRV Env (fig. S4B). XMRV protein expression in CFS patient–derived activated T and B cells grown for 42 days in culture was confirmed by Western blots (fig. S4C) using Abs to SFFV Env and xenotropic MLV.

We next investigated whether the viral proteins detected in PBMCs from CFS patients represent infectious XMRV. Activated lymphocytes (6) were cocultured with LNCaP, a prostate cancer cell line with defects in both the JAK-STAT and RNase L pathways (10, 11) that was previously shown to be permissive for XMRV infection (12). After coculture with activated PBMCs from CFS patients, LNCaP cells expressed XMRV Env and multiple XMRV Gag proteins when analyzed by Western blot (Fig. 3A) and IFC (fig. S5A). Transmission electron microscopy (EM) of the infected LNCaP cells (Fig. 3B), as well as virus preparations from these cells (Fig. 3C), revealed 90- to 100-nm-diameter budding particles consistent with a gamma (type C) retrovirus (13).

We also found that XMRV could be transmitted from CFS patient plasma to LNCaP cells when we applied a virus centrifugation protocol to enhance infectivity (6, 14, 15). Both XMRV gp70 Env and p30 Gag were abundantly expressed in LNCaP cells incubated with plasma samples from 10 of 12 CFS patients, whereas no viral protein expression was detected in LNCaP cells incubated with plasma samples from 12 healthy donors (Fig. 4A). Likewise, LNCaP cells incubated with patient plasma tested positive for XMRV p30 Gag in IFC assays (fig. S5B). We also observed cell-free transmission of XMRV from the PBMCs of CFS patients to the T cell line SupT1 (Fig. 4B) and both primary and secondary transmission of cell-free virus from the activated T cells of CFS patients to normal T cell cultures (Fig. 4C). Together, these results suggest that both cell-associated and cell-free transmission of CFS-associated XMRV are possible.

Topic: Xenotropic Murine Leukemia Virus-Related Virus (XMRV) – Informational Presentation

Issue: FDA is updating the committee on XMRV, the newly identified human retrovirus (a gammaretrovirus), and FDA’s collaborative efforts with other public health agencies and blood establishments to determine whether XMRV poses a safety concern for the blood supply.

Background:

Xenotropic Murine Leukemia Virus-Related Virus (XMRV) is a newly identified retrovirus and the first gammaretrovirus of the Retroviridae family detected in humans. It is unrelated to HIV but highly similar to mouse retroviruses. Gammaretroviruses are widespread in vertebrates, but do not establish infection readily in immunocompetent hosts. Gammaretroviruses in animals are implicated in a wide range of diseases including cancers, immunodeficiency, and neurological disorders. XMRV is a simple retrovirus with gag, pol and env genes and unlike HIV or HTLV, has no accessory or regulatory genes. The genome is a dimer of linear, positive-sense, single stranded RNA about 8,300 nucleotides long.

XMRV was identified using a viral detection DNA microarray composed of oligonucleotides corresponding to the most conserved sequences of all known viruses (1). Using this array, gammaretroviral sequences were identified in samples from 7/11 prostate cancer (PC) patients that were homozygous (QQ) for the R462 mutation for RNAse L which is an important molecule in the innate antiviral response (2). A survey of an additional 86 PC tumors by RT-PCR specific for XMRV showed that 40% of QQ cases were positive for XMRV compared with 1.5% of heterozygous RQ and homozygous wild type RR cases. Subsequently an analysis of 334 consecutive prostate resection specimens using a quantitative PCR assay and immunohistochemistry showed that 6% were positive for XMRV DNA and 23% for protein expression. Taken together, these findings suggest an association of XMRV with prostate cancer (3).

A subsequent study showed that XMRV could also be detected in patients with Chronic Fatigue Syndrome (CFS), a disease of unknown etiology, known to affect several million people worldwide (4). DNA from peripheral blood mononuclear cells (PBMCs) of CFS patients and healthy controls was tested for the presence of XMRV sequences. XMRV sequences were found in 68 out of 101 CFS patients (67%), compared with 8 of 218 (3.7%) healthy US controls. The viral gene sequences identified in CFS patients clustered with sequences from PC and both sequences were virtually identical. Further investigation using activated CFS patient PBMC co-cultured with susceptible PC cells (LNCaP) showed that virus could be transmitted by cells and supernatant, as indicated by protein expression and transmission electron microscopy, suggesting that the virus being detected by protein expression was infectious. Virus could also be transmitted from 10/12 CFS patient plasma samples. Taken together, these studies suggested that both cell associated and cell-free transmission of XMRV is possible. Finally, antibodies to XMRV were detected in 9/18 CFS patients using a test based on the envelope of a closely related virus, spleen focus forming virus (SFFV).

Preliminary data from a limited study of XMRV in an animal model (rhesus macaques) showed disseminated infection but only low but detectable transient viremia between 4-14 days. Seroconversion occurred at 11-14 days following inoculation, with titers peaking around day 95. Virus was isolated from T- and B- lymphocytes and NK cells in blood (but not macrophages), prostate epithelial cells, vaginal tissue, and there was evidence for viral replication in spleen, lung, lymph nodes and liver (5).

Since the discovery of XMRV there have been several controversial reports about the association of XMRV with PC and CFS. Studies of an Irish PC cohort of 139 patients with the R462Q mutation showed no evidence of XMRV (6). In a German study, only 1/105 tissue samples were positive for XMRV DNA from non-familial PC while 1/70 samples were positive from men who did not have PC (7). In a different study of German PC patients, 589 PC tissues with the RNaseL R462Q mutation were tested using nested RT-PCR and all were found to be negative (8).

In regard to CFS, several recent studies have reported negative findings of XMRV in blood. A Dutch study failed to detect XMRV in PBMC of patients with CFS (9) and similar findings were reported in two studies from the UK although a few samples showed XMRV neutralizing activity in one study (10,11). Studies conducted by CDC scientists in a US population of CFS cases and blood donors showed no evidence of XMRV infection in PBMC and plasma. These data do not lend support to an association of XMRV with CFS or its presence in blood donors. (13)

Among other related findings are those recently reported by investigators in Germany that XMRV could be detected in respiratory secretions (14). In this study, 267 respiratory samples taken from German patients were screened for XMRV infection by PCR assay. The prevalence of XMRV DNA was 2.3% (3/75) in travelers from Asia who had respiratory tract infections; 3.2% (1/31) in patients with chronic obstructive pulmonary disease; 9.9% (16/161) in immunosuppressed patients with severe respiratory tract infections; 3.2% (2/62) in the healthy control group. It is not clear whether finding XMRV in the respiratory tract indicates that the virus can be transmitted by the respiratory route (14). In general, retroviruses, like HIV-1, are rarely transmitted through respiratory secretions.

A compilation of all the XMRV studies done so far is shown in appendix I. The reasons for discrepancies between findings are not clear. Differences in cohorts, populations and test methods have been considered as potential causes. In addition, differences in geographic prevalence, genetic variation of the virus or other factors could also potentially contribute to these conflicting findings. Additional studies are needed to confirm disease association of XMRV and to fully understand the role of XMRV in disease causation.

Discussion:

(a) Detection methods:

Currently there are no commercially approved/licensed tests for detection of XMRV infection. The assays used for research studies include PCR, EIA, Western blot, and immunohistochemical methods. The relative sensitivity and specificity of various assays have not been determined and standards for performance of assays have not yet been established.

(b) Evaluation of Transfusion Risk in the US:

Transmission through transfusion has not been shown, but is theoretically possible since virus has been detected in blood cells and there is evidence of cell-free virus. A seroprevalence study in Japan found 1.7% of random donors to be positive (12). Preliminary studies have shown that 3/2851 US blood donors (0.1%) were anti-XMRV antibody positive using a research immunoassay based on gag and env proteins (5).

A Blood XMRV Scientific Research Working Group, led by the National Heart Lung and Blood Institute (NHLBI), has been established to identify and design research studies to evaluate whether XMRV poses a threat to blood safety. An evaluation of blood safety risks includes 1) an evaluation of the prevalence of XMRV in blood donors; 2) if prevalent in blood donors, a determination of whether the virus is transfusion-transmitted; and 3) if transfusion-transmitted, whether clinical manifestations occur. An evaluation of the prevalence of XMRV in blood donors necessitates a way to measure or identify the agent in blood specimens. The fact that different laboratories obtain different results for XMRV detection in patients’ specimens and different prevalence estimates in normal controls strongly suggests the need for the development of standard XMRV reference and clinical case and healthy donor prevalence panels for XMRV detection. Thus, development of XMRV nucleic acid test (NAT) analytical and clinical panels was identified as the first priority by the Working Group. As a first step towards this goal, an analytical reference panel was developed to validate NAT assays for use in donor survey studies . The panel was developed by the Blood Systems Research Institute which is the Central Laboratory for the NHLBI Retrovirus Epidemiology Donor Study-II (REDS-II). Virus stocks were prepared from culture supernatants of the chronically infected prostate cancer cell line, 22Rv1. Following titration, reference infected cell preparations were established by spiking known numbers of infected cells into whole blood. Similarly, reference infected plasma preparations were established by spiking pedigreed-negative plasma with known viral titers of cell-free culture supernatants at serial dilutions. These coded analytical reference panels of specimens containing varying concentrations of XMRV virus as well as clinical panels which include biospecimens from blood donors, patients with Chronic Fatigue Syndrome whose blood was previously found to be positive for XMRV by the Whittemore Peterson Institute, and positive and negative control specimens are in the process of being tested by up to six participating laboratories to compare the sensitivity of their respective assays for XMRV. The study participants include laboratories from the NCI, FDA (two laboratories), CDC, Blood Systems Research Institute and theWhittemore Peterson Institute.

Summary:

The BPAC session will include several informational presentations on XMRV including a background and review of studies that led to the identification of the virus, ongoing validation studies of XMRV panels, updates of research efforts on test/panel development and other studies.

Finally, FDA will periodically update the committee on progress in our understanding of XMRV and transfusion safety. As additional data are obtained on prevalence in blood donors and any associated transfusion risks, BPAC may be asked to advise FDA on appropriate measures to maintain the safety and availability of the blood supply.