Case study: Disminution of plasma viral load and cultured HIV-infected peripheral blood mononuclear cells

HIV AIDS Rev, 2004;

Disminution of plasma viral load and cultured HIV-infected peripheral blood mononuclear cells in non-responding patients treated with two calf thymus nuclear proteins and conventional antiretrovirals.

J. Jacobo Ayala-Gaytán1, Eduardo R. Zapata de la Garza1, Salvador B. Valdovinos-Chávez2, Leticia Navarro-Marmolejo3, Javier Vargas Villarreal3,Herminia G. Martínez-Rodríguez4, Rebeca Palacios-Corona3, Ricardo M. Cerda-Flores5, Hampar Karageosian6, Harry Zhabilov6, Patia Arnavdora6, Salvador Said-Fernández3

1Servicio de Infectología, Hospital Regional de Especialidades 25, Instituto Mexicano del seguro Social (IMSS).
2Coordinación Delegacional de Investigación en Salud, IMSS. Delegación Regional Nuevo León.
3División de Biología Celular y Molecular, Centro de Investigación Biomédica del Noreste (CIBIN). IMSS. Ave. 2 de Abril y San Luis Potosí, Col. Independencia, 64 720 Monterrey, N.L. México.
4Unidad de Laboratorios de Ingeniería y Expresión Genética. Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, N.L.México.
5 División de Genética, CIBIN, IMSS.
6 Viral Genetics Inc. 905 Mission Street, South Pasadena, California 91030, USA.

Summary:Background:Multidrug-resistant type 1 human immunodeficiency virus (HIV-1) variants rise in antiretroviral-exposed patients, and new therapeutic strategies are needed. We evaluated the efficacy of two thymus nuclear proteins (TNP), used in combination with antiretroviral medications, in 10 patients with intermediate and advanced HIV infection.

Material/Methods: Ten HIV-positive volunteers received personalized antiviral therapy along with TNP (i.m.,
twice weekly for eight weeks). Disease progression was evaluated in each patient, clinically and by laboratory tests. Variables were assessed with repeated-measure analysis.

Results: The baseline viral load was 3.32–4.6 log10/mL. Within 1–3 months after the first injection, these values decreased in five patients to undetectable levels (HIV-1 RNA < 40 copies/mL, 50% efficacy), and remained in this condition until the sixth month. The proportion of HIVfree peripheral blood mononuclear cells (PBMC) at quantitative culture increased 11 and 22-fold with respect to baseline. The quantitative PBMC measurement reached 100% efficacy (P<0.0001) in raising the number of HIV-uninfected PBMC/1 HIV-infective particle with respect to baseline. No serious adverse events were observed.

Conclusions: TNP administration safely restores the efficacy of conventional antiretroviral strategies for at least six months, possibly by blocking the entry of HIV-1 to the target cells.
Key words: AIDS•HIV-1 infection•Thymus nuclear proteins•Viral load•PBMC• combined antiretroviral therapy•Calf thymus nuclear proteins.

Author’s address: Salvador Said-Fernández, Ph.D. División de Biología Celular y Molecular, Centro de Investigación Biomédica del Noreste (CIBIN). IMSS Delegación Regional Nuevo León, Ave. 2 de Abril y San Luis Potosí, Col. Independencia, 64 720 Monterrey, N.L. e-mail: salvador.said@mail.mty.itesm.mx. phone/fax: (01152-81) 8190-4035.

BACKGROUND
AIDS is global and its incidence and mortality have risen systematically [1]. Since 1950, more than 60 million people have been infected with the virus, causing 22 million deaths. In 2001, an estimated 40 million people globally were living with HIV, and 14,000 persons were infected daily [2]. This pandemia also plays a determinant role in the re-emergence of tuberculosis [3]. Highly active antiretroviral therapy (HAART) diminishes the viral burden to undetectable values in 2–4 months [4]. Few years ago, Ho [5] suggested that eradication of HIV from a human required effective viral suppression for more than 10 years in patients bearing large viral pools.

Recent studies consider this task to be more difficult than previously thought [6], mainly because the available antiretrovirals are significantly toxic [7], latently infected immune cells show prolonged viability [4,5,6,8], and HIV has the capacity to produce multidrug-resistant (MDR) variants [9,10], compelling clinicians to change the therapeutic strategy periodically until no alternatives remain [11].

Accordingly, there is the continuous need to seek for new and more potent combinations of effective antiretroviral medications [10]. Two Calf thymus nuclear proteins (TNP), weighing 12.5 kDa and 14.5 kDa, strongly bind in vitro to the HIV transmembrane glycoprotein gp41 (Fig. 1), suggesting a possible role in inhibiting the fusion of HIV to specific target cells.

To endorse such an assumption, we found that virion particles actively infected peripheral blood mononuclear cells (PBMC) from healthy individuals when co-cultivated with cells from HIV-infected patients, but failed to do so when PBMC from infected patients were pre incubated with TNP (unpublished data). In pre-clinical studies, TNP was demonstrated as safe for mice and rats, at escalating intramuscular doses from 0.1 to 0.5 mg/kg of body weight; or 1.24 mg/kg when administered to rabbits twice weekly, for eight weeks (unpublished data). The objective of this pilot study was to analyze the ability of TNP administration to restore the efficacy of combined antiretroviral therapy.

MATERIAL AND METHODS
Study design. This was a pilot two-sectional study, with cuts at three and six months after the first intramuscular TNP injection. Patients. Ten HIV-infected adult male volunteers were recruited from the outpatient infectious diseases service at IMSS Hospital Regional de Especialidades 25 in the city of Monterrey, Mexico. Three individuals were at an intermediate, and seven at an advanced stage of the disease. They were failing to respond to the second or third HAART regimen. At the start of the study, the patients had been infected for between six months and six years. Treatment and management of patients. The protocol of the present study was approved by the review and ethics board of IMSS-Hospital Regional de Especialidades 25 and of the Mexican Health Secretariat. All participants gave written informed consent. They received antiviral nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitor (PI) combinations before, during and at least one year after the application of TNP therapy. This medication was developed by Viral Genetics Inc., Pasadena, CA, USA) and its safety and clinical efficacy is currently being evaluated, under the (USA) registered brand “VG-100”. TNP was administered on a compassionate basis. Patients received 6 mg per ml i.m. injections every Wednesday and Thursday for eight consecutive weeks. Data relative to periodical clinical evaluations were collected every two weeks during the first three months, and six months after the first TNP application. Individual questionnaires for general health status, reporting either undesirable side effects of TNP, or the emergence of serious adverse events attributable to the experimental medication, were completed each time. At every evaluation visit, blood samples were collected to determine chemistry profile, blood count, CD4+ thymocyte count, plasma HIV-1 RNA levels and HIV-1 quantitative PBMC cultures. Laboratory Methods. Binding electrophoresis studies were performed in agar gels, which were cast on 75 × 75 mm glass plates with 14.5 ml of 1% melted agarose in electrophoresis buffer (Tris base 2.42 g/l, thiodiglycol 1.2 ml/l v/v, calcium lactate 0.109 g/l, EDTA 0.35 g/l, sodium tetraborate decahydrate 3.81 g/l, glycine 3.75 g/l, pH 7.6). Wells 1 and 3 were loaded with 7 μL of 0.8 mg TNP/ml plus 6 μl of 1 mg gp41 fragment 579–601/ml; holes 2 and 4 with 7 μl TNP, and well 5 with 6 μl gp41 fragment. These preparations were incubated at room temperature for 10 min, the electrophoresis chamber was loaded with seam buffer. Electrophoresis was run at 165 V, 80 mA for 45 min. The gels were fixed with a ethanol:acetic acid (9:1). The proteins in the gel were stained with 0.5% Brilliant blue R 250 in ethanol:acetic acid:water (4.5:1:5), washed with ethanol:acetic acid (9:1), and dried. Salts and gp41 fragment were purchased from Sigma Chemical Co. (St. Louis MO). Plasma viral load was determined as the number of RNA copies/ml by means of reverse transcription polymerase Figure 1. Binding of TNP to gp41. Wells 1 and 2 were loaded with 7 μl TNP plus 6 μl gp41; wells 3 and 4 with 7 μl TNP alone, and hole 5 with 6 μl gp41. Symbols indicate the position of the anode (-) and cathode (+). Note that the gp41 fragment alone migrated toward the anode, but when TNP was present, it moved toward the cathode.
1
2
(–) 3
4
5
(+)

chain reaction (RT-PCR: Ultra Sensitive RT-PCR Amplicor system, Roche Diagnostics Co. Indianapolis, Indianapolis), with a sensitivity range from 40 (1.6021 log10) to 40,000 (4.6020 log10) HIV-1 RNA copies/ml of plasma. The ratio of cultured PBMC bearing at least one infective viral particle to the number of non-infected PBMC (expressed as 1 HIV-1 infective particle/the number of non-infected PBMC) was determined in vitro cultivated PBMC from each patient [4]. Sensitivity range limits for this method were from 320 to 1×106 PBMC/1 infective viral particles [12]. The count of CD4+ thymocytes in peripheral blood was determined using monoclonal anti-CD4 antibodies labeled with fluorescein isothiocyanate. Counts of fluorescent cells were determined by flux cytometry [12]. Blood samples were drawn for baseline before starting TNP injections and at three and six months later. T-cell counts and HIV-specific tests were performed by Specialty Laboratories, (Santa Monica, CA).

Routine blood counts and chemistry profiles were analyzed at local hospital facilities. Adverse effects criteria. Serious adverse events to be recorded in this trial included death, or life-threatening events; appearance of organic dysfunction or impairment; any clinical condition requiring hospitalization; and any neoplasm different from those associated with HIV infection.

Statistical analysis. Sample size was calculated for a repeated-measurement experimental design analysis (ANOVA), fixing two-tailed α = 0.05 and one-tailed β =
0.20 for each clinical test [13]. For statistical purposes SPSS-PC V10.0 software (SPSS Inc., Chicago, IL) was used. A value of p < 0.05 was considered significant.

RESULTS
Basic studies: binding of TNP to gp41
Figure 1 shows that the gp41fragment and TNP migrated in opposite ways when they were electrophoresed separately, gp41 to the anode and TNP to the cathode, while the TNP and gp41 together migrated to the cathode.
Body weight
The baseline mean ± SD body weight was 68.2 ± 7.88 kg, and 3 and 6 months after the first TNP application it was 69.38 ± 7.35 and 68.3 ± 7.25 kg, respectively. No significant changes were observed during the trial (F = 0.12, p = 0.18). In the sixth month, four of the patients had gained 0.5–5.0 kg, five had lost 0.6–2.1 kg and one did not show any
changes with respect to baseline.

CD4+ Thymocyte counts
After three months, the number of CD4+ thymocytes increased by 2.6–12.2% in seven patients, and in three the counts diminished by 1–7.6% with respect to baseline. In the sixth month, the CD4+ thymocyte counts in seven patients were 21% lower than baseline ones. The mean (± SD) T CD4+ thymocyte counts at the beginning of the study, and at the third and sixth month were 133.5 ± 78.1, 147.7 ± 82.1, and 117.1 ± 75.5, respectively. The changes observed after six months were significant (p = 0.016)
(Table 1).

Plasma HIV-1 RNA
In eight of the patients, the number of HIV-1 RNA copies in plasma increased to 0.1–1.5 log10/ml between the second and eighth weeks. This parameter then descended systematically (data not shown). At the third month, the plasma viral burden had diminished (1.72 to 2.68 Log10/ml) in five patients to undetectable levels; in two patients viral burden diminished by 0.05 and 0.09 log10/ml, respectively; in two this parameter increased by 7–11%, and no changes were observed in one. The changes observed with respect to the baseline were highly significant. The efficacy (number of participants/number of patients whose plasma burden became undetectable) of the experimental treatment was 50% from the third month. In three patients, an additional diminution (0.03 log10) was observed at six months with respect to viral load that they had at the third month. The average ± SD of log10 RNA HIV-1 particles of the entire
Table 1. CD4+ thymocyte count.
Number of CD4+ thymocytes/ml
Patient Basal At 3rd At 6th

ANOVA: F = 7.94, p = 0.003: Basal ≠ 3rd month=6th month
group was 25% and 28% lower than that of baseline at third and sixth months, respectively (Table 2). Quantification of HIV-1 in PBMC culture At the third month of the experimental treatment, the number of cultured PBMC free of HIV-1 increased in nine patients, and four achieved the limit of detection ( 1 PBMC bearing at least 1 VIH-1 particle/1 × 106 healthy PBMC). At the sixth month, all reached undetectable levels in cultured PBMC. Furthermore, at the third month three of these patients presented with both undetectable plasma HIV-1 RNA and HIV-1 in cultured PBMC; and at the sixth month two more patients also achieved this condition. In the entire group, the mean (± SD) baseline value of HIV-1-free PBMC was 45,120 ± 57,068; and at the third and sixth months, 498,000 ± 387,751, and 1,000,000 ± 0.0, respectively. Thus, patients increased the proportion of non-infected PBMC 11–22-fold with respect to the baseline at the third and sixth months, respectively (p = 0.0001: Table 3).
Other laboratory tests
Table 4 shows that, with the exception of L-alanine aminotransferase (ALT), no significant changes in biochemistry were observed during the study.
Serious events or severe adverse side effects
None of the ten volunteer patients experienced any serious events or severe side effects during the treatment or within one year after it was started.
Non-serious side effects
During the study, six patients developed mild folliculitis that was easily controlled with antimicrobial drugs. The patients experienced some discomfort, mainly during the first two weeks after starting TNP injections. Beyond this period, many of the symptoms strikingly diminished or ceased. Complaints were as follows, and in no case deserved hospitalization: pain at the injection site (episodes lasting 3–6 days); insomnia; fatigue; dizziness observed immediately after the injection of TNP (one patient suffered transient syncopes); watery diarrhea (appearing before the decline of viremia); headache; anorexia; and low back pain.

DISCUSSION
TNP injections, when associated with conventional antiretroviral therapy, did not produce any serious adverse events in our patients. Two years after the conclusion of our study, all the patients were alive and receiving antiviral medication, with variable degrees of disease progression. The only clinical data suggesting an unfavorable response to the experimental treatment was a 27% increase in ALT levels with respect to the baseline.These changes were clearly attributable to the antiretroviral medication [14]. The two main outcomes observed in the studied group of patients were the decrease of plasma HIV-1 RNA levels, and the increased proportion of HIV-1-free cultured PBMC. Reduction of viral load by 0.5 log10 within the first four weeks, leading to undetectable viral loads four to six months after the beginning of the treatment, are highly suitable criteria for decisions to change the regimens. These are widely accepted measures of success when assessing therapeutic strategies for controlling the progression of HIV infection [15], and for establishing the prognosis: the greater and faster the diminution of viral load, the better the prognosis, leading to prolonged survival and better quality of life [16,17].
By the third month, viremia became undetectable in five previously refractory patients (50%), and in three there was an additional diminution in their viral load at six months. The maximum favorable response appeared two to eight weeks after the first TNP injection (data not shown). Thus, the apparent control of viral replication is not synchronous, and suggests that this response depends upon individual characteristics: possibly the stage of the disease, physical status of patients, and viral pool sizes [5]. Two of our patients started the trial with HIV-1 RNA levels beyond the upper limit of quantification (4.6020 log10). In general, the lower the initial viral load, the quicker the diminution after initiation of the experimental treatment.

It is noteworthy that at the sixth month all patients achieved undetectable values of infected PBMC cells by quantitative culture. Detection of HIV-1 infective particles is considered a more stringent criterion than plasma viral load for assessing the infective capacity of HIV-1 [4, 8,18–20]. This is because plasma HIV-1 RNA quantification by PCR does not discriminate between infective viruses and non-infective viral RNA fragments [17–21]. We hypothesize that the diminution of infected PBMC and viral load to undetectable levels occurs because TNP possesses a strong in vitro affinity to bind gp41, as shown here. This suggests that TNP could prevent the fusion of HIV-1 to the surface of the target cells [2,22–24], as do other agents, such as T-20 and T-1249, that block virus cell fusion by binding to the viral envelope glycoprotein gp41 [25]. It has been pointed out that after the control of viral replication is achieved, the drop of plasma viral load in a patient takes from 40 days [26] to 4 months [27], and so HIV-1 reduction to undetectable levels in cultured PBMC indicates an effective control of viral replication. We also found that the mean number of CD4+ T cells reduced slightly, but significantly at the third month, and the viral load increased during the first eight weeks. A possible explanation could be that reactivated CD8+ T cells in our patients could trigger the lysis of infected cells. Accordingly, TNP may have an effect similar to other antiviral agents, such as interleukin-2, which modulate the cells’ immune response [28,29], Although this group of participant volunteers was small, we found statistically significant improvements in two important markers (viral load and rate of PBMC/1 harboring infective viral fragments). Furthermore, our patients had been suffering from advanced stages of their disease, and conventional antiretroviral strategies applied to them were failing until we used TNP. This is a preliminary study, and it shows that TNP therapy may be safely associated with other antiretroviral therapies, and may therefore have value in promoting the available therapeutic combinations when antiretroviral drugs fail. The availability of complementary medications such as TNP is desirable, considering that some 30% of patients fail to suppress their HIV infection despite the use of potent combined therapy, and that viral rebound rates after initial viral load suppression to undetectable levels (secondary failure) typically range between 40% and 60% after one year from initiation of HAART. In addition, nearly half of patients with HIV infection carry multidrug-resistant variants [8]. Combining antiretroviral medication with TNP thus appears to be effective to achieve viral control in HIV-infected patients. Further studies, addressed to determine the number of PBMC bearing proviral DNA and the persistence of HIV-1 transcription in these cells, should be carried out. A longer period of observation is also needed to determine if plasma viral load will fall below threshold levels in patients having an undetectable number of infected PBMC, and to assess the length of time in which these changes takes place.

A phase II trial is clearly warranted.

ACKNOWLEDGMENTS
We thank for the technical assistance of Victor R. Rosales- García, MD, Susana Manriquez-Lomas, RN, and the laboratory personnel from the Hospital Regional de
Especialidades 25, IMSS, Monterrey, N. L. México.

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