|Mucosa; Therapeutic vaccine; Immunomodulation; HCV;
|Hepatitis C virus (HCV) is a global public health problem, affecting
an estimated 180 million people . The current standards of care
are interferon alpha or pegylated interferons alone or in combination
with ribavirin. However, they have shown limited success and are
associated with undesirable side effects [1,2]. The high cost of
medications is another barrier that prevents wider use of anti-HCV
therapy, especially in developing countries. Thus, the treatment
of chronic HCV infection represents an unmet medical need. This
problem is further aggravated when patients are co-infected with
TB and/or HIV. It is agreed that the resurgence in the incidence of
global TB is due in part to the AIDS epidemic. There are 9.2 million
new cases and 1.7 million deaths from TB annually. According to
various sources between one-third and one-half of patients with TB
are infected with HIV, which is associated with very poor prognosis
and high mortality. Despite the overwhelming burden of disease,
no new anti-TB compounds were developed in last 40 years and
current strains of TB are becoming increasingly resistant to existing
drugs. The occurrence of chronic hepatitis C along with TB and HIV
in the same patient presents a challenging task with practically no
therapeutic solution . One of promising treatment approaches
being pursued by many investigators around the world is so-called
therapeutic vaccination, whereby the immune system of a host is
modulated to fight off an infectious pathogen .
|In previous studies, conducted in Mongolia, we have shown
that oral therapeutic hepatitis vaccine V-5 Immunitor (V5), was
beneficial to patients with hepatitis B and hepatitis C [4,5]. The
anecdotal evidence that became apparent during these investigations suggested that V5 could be beneficial to TB patients as well. In order
to confirm these observations we have undertaken an independent
study in Ukraine by recruiting patients with chronic hepatitis C.
Due to fortuitous circumstances this study was carried out at a TB
hospital with extensive experience in TB immunotherapy and hence
the enrolled patients had concomitant Mycobacterium tuberculosis
infection [6-8]. Furthermore, all individuals in this patient population
were infected with HIV, which is common among individuals who
acquire these infections through needle sharing practice. Neither
AIDS therapy, nor HIV monitoring was carried out during this study
since patients hospitalized in TB dispensaries in Ukraine are cared
for TB only. Thus, even though our study was aimed at evaluating the
effect of V5 in HCV patients it happened that they were also treated
with standard anti-TB drugs (ATT).
|Materials and Methods
|Five female and 15 male patients with chronic hepatitis C infection were enrolled into open-label, one-month study. After
ethical approval by the internal review board of the Lisichansk TB
Dispensary, patients who had higher than normal baseline liver
enzyme transaminase ALT and bilirubin levels were enrolled into
the trial. All patients were hospitalized and presented with fever
and cough and other common TB symptoms such as chest pain,
dyspnea, hemoptysis, weight loss and anorexia. The median age
of patients was 34.5 years, range 24-74 years, mean±SD equal to
36.1±10.6 years. All patients were positive for hepatitis C antibodies
and anti-HIV antibodies as well (ACON Biotech, Hangzhou, PRC).
The number of patients with first diagnosed TB, HCV, and HIV was
8, 5, and 8 respectively. The average/median duration of TB, HCV,
and HIV infections prior to study initiation was 3.9/4, 2.2/2 and 2.5/2 years respectively (Figure 1). None of the patients were treated with
interferon and/or anti-hepatitis drugs before or during the trial.
Patients received standard or individualized ATT but no HIV therapy.
|V-5 Immunitor (V5)
|V5 is derived from pooled blood of hepatitis B and C carriers by
employing proprietary technology developed by us. The process of
manufacturing is described in detail earlier  – it involves heat- and
chemical inactivation with subsequent formulation into a tablet. The
principle for production of V5 is not much different from established principles with old-fashioned killed vaccines, e.g., Hepatitis B vaccine
made from pooled plasma. V5 is currently approved in Ukraine as an
immunomodulating supplement. V-5 Immunitor is presented as an
850 mg coated pill, ten of which are sealed in a “blister” pallet, with
30 pills per one package. The recommended dose is one to two pills
per day. The preparation is stable at ambient temperature for five
|Administration schedule and monitoring
|The patients with confirmed pulmonary TB were treated with
WHO-recommended regimen of antibiotics, i.e., Izoniazid (H) 300
mg; Rifampicin (R) 600 mg; Pyrazinamide (Z) 2,000 mg; Streptomycin
(S) 1,000 mg; and Ethambutol (E) 1,200 mg in combination with one
daily tablet of V5, which was given half-an-hour before or after the
morning meal. The treatment was administered in an inpatient setting
to patients hospitalized in our dispensary. The baseline and outcome
parameters were established at study entry and at the end of followup.
The ALT and other biochemical values were measured by LiquiUV
test (Human GmbH., Germany). Rapid test kits for anti-HCV and HIV
antibodies were from ACON Biotech (Hangzhou, PRC). In addition
to clinical and biochemistry evaluations a standard microbiology
examination of sputum smear staining by Ziehl Neelsen method was
conducted prior to study entry and at post-treatment period.
|Primary endpoints for this study were changes in serum ALT
transaminase, total bilirubin, liver size, and clinical response.
Secondary endpoints were parameters related to TB infection.
Parametric values were assessed by paired Student t-test and
qualitative changes were analyzed by Fisher’s two-way contingency
table using internet-based free software (GraphPad Software, Inc., La
Jolla, CA). The significance level was set at P≤0.05.
|The results of adjunct V5 immunotherapy which lasted one
month are shown in Table 1. The statistical values from paired
t-tests, provided at the bottom of the Table 1, show that V5 produced
significant positive changes. Without exception every endpoint of the
study had reached highly significant statistical P value compared to baseline, indicating that these parameters were correlated with each
other and associated with better clinical outcome.
|The beneficial effect of V5 on hepatitis is supported by all three
liver damage endpoints including normalization in ALT and bilirubin
levels and decrease in abnormal liver size. Indeed the observed
reduction was highly significant, from 1.72±0.34 to 0.18±0.3 µmol/
ml•h (P=5.0 E-012) and 22.1±3.4 to 10.9±2.5 µmol/L (P=5.7 E-009)
for ALT and total bilirubin respectively. All, except one patient, noted
disappearance of pain upon liver palpation. Enlarged liver reduced
from 3.5±1.4 to 0.95±1.1 cm above normal size (P=2.9 E-009). This
occurred despite the concurrent treatment of patients with anti-
TB drugs which are known to produce the adverse effect on liver
|In addition, patients experienced remarkable amelioration in TBassociated
inflammation indicators such as erythrocyte sedimentation
rate, elevated leukocyte counts and persistent fever. After one
month on V5 the erythrocyte sedimentation rate and leukocyte
counts returned back to normal from 32.3±11.4 to 9.9±6.4 mm/h
(P=3.7 E-008) and 14.3±3.9 to 4.7±1.4 x 109 cells/L (P=7.1 E-010)
respectively. Remarkably, TB-associated fever subsided in 19 out
of 20 patients within mean/median 4.1/3 days (Table 2). This was
most likely due to V5 intervention since patients were on ATT for an
average 3 months prior to the immunotherapy and despite that they
had persistent above normal temperature.
|Sputum smear examinations revealed that V5 appeared to
accelerate the conversion rate. Out of 17 patients who were positive
at baseline 16 individuals (94%) became smear-negative after one
month of adjunct immunotherapy. The only patient (#7) who failed
to convert had also exhibited poor response profile as judged by
separate lab and clinical parameters. Other TB-associated symptoms
such as anemia and weight loss have improved as a result of V5
intervention. The hemoglobin content increased from 114±7.1 to
123.4±6.6 g/L (P=1.4 E-007) and average body weight gain was 7.7
kg (P=4.6 E-007), range 4-13 kg.
|The most remarkable finding from the patients’ perspective is the
disappearance of baseline clinical symptoms after one month on V5
immunotherapy. Improved patients’ well-being such as better quality
of life, weight gain, increased appetite, enhanced physical strength,
better mood, and other subjective parameters have been undeniable
but were difficult to assess objectively. Only one patient (#7), failed
to respond fully to the therapy and this impression is corroborated by
clinical and laboratory data. Based on sputum conversion and clinical
improvement 17 out 20 patients were discharged from the hospital
after one month and remaining three continued receiving further
|Open-label, one-month, phase 2 trial of V5 administered orally
once per day revealed normalization of biochemical markers of
hepatitis and remarkable clinical improvement among 19 out of 20
patients with chronic hepatitis C. These findings support our earlier
clinical investigations in two unrelated chronic hepatitis diseases,
namely hepatitis B and C [4,5]. V5 was found to be safe and had
reversed the signs of liver damage. This observation is significant
since anti-TB drugs are prone to cause hepatotoxicity, especially
among those with chronic hepatitis . Thus, the hepatoprotective
property of V5 could be advantageous even to those individuals who
are treated with ATT, but do not have the underlying viral hepatitis.
From prior experience with V5 we know that HCV viral burden can be reduced as a result of therapeutic vaccination . However, due
to financial constraints we were not able to measure HCV RNA in
this study and thus we do not have information about the effect of
V5 on viral load in this population of patients. We need to conduct
additional studies to find this out.
|Several therapeutic vaccination strategies are now being
explored for hepatitis C . These include DNA immunization,
peptide-based vaccines, plant-expressed vaccines, virus-like particles,
and presentation of HCV antigens via dendritic cells. In addition to
academic and non-profit institutions several biotech companies are
involved in the development of therapeutic HCV vaccines. Most
vaccines from these companies are in the preclinical stage. A few
vaccines like TG4040 (Transgene); PeviPRO (Pevion); ChronVac-C
(Inovio/Tripep); GI-5005 (GlobeImmune); and HCV/MF59 (Chiron/
Norvartis) have advanced into safety phase 1 stage. Only three
vaccines have been tested in advanced efficacy trials but have failed
to reach study endpoints and were subsequently abandoned . The
therapeutic vaccine most relevant to this study is an oral preparation
made from protein extract of human hepatocytes mixed with NS3
protein of HCV. This vaccine, which was developed by Yaron Ilan
and his team with support of ENZO company, has shown good safety
profile in phase 1 trial .
|The postulated mechanism of Ilan et al., vaccine is similar to that
of V5. The mucosal or oral administration of viral antigens entails
a complex immune tolerance state, characterized by simultaneous
enhancement and suppression of different elements of the immune
response in a manner that benefits the host. Such a manipulation of the
mucosal immune response against viruses may achieve a combination
of specific anti-viral immunity and inhibition of immune-mediated
liver injury. Since HCV is not cytopathogenic very little is known as
to how the liver becomes damaged as a result of viral infection .
We subscribe to the theory that progressive liver injury is caused by
self-directed immune reaction of certain T cell subsets against virusinfected
hepatocytes. Although the autoimmune nature of liver injury
has been recognized by some investigators, others maintained that
virus-induced autoimmune reaction in hepatitis is a rare event .
Nevertheless, it is clear that elevated serum aminotransferases and
other hepatic injury markers, e.g., bilirubin, are definitively the signs
of ongoing inflammatory liver damage associated with HCV.
|In our opinion, a similar concept is applicable to TB, whereby
pulmonary or other tissues harboring mycobacteria are constantly
assaulted by host’s immune system, creating an inflammatory state
and tissue damage akin to hepatitis. Thus, in order to treat these
diseases one needs to induce specific immune tolerance rather than
overdriving an already intense immune response . However,
there are practically no clinical studies that have addressed the
immunological basis of TB infection from such a viewpoint .
Immunosuppressive regimens like adjunctive use of corticosteroids
or TNF blockers have produced conflicting results suggesting that
wholesale suppression of the immunity is fraught with risks of
exacerbating the disease [12-15].
|Currently available TB chemotherapy is not perfect; it requires
several TB drugs to be taken in combination for long periods of time
[6-8]. This can cause multiple side effects, poor drug adherence,
treatment failure, and the emergence of drug resistance with
major social and economic consequences, especially in low-income
countries. It is agreed that novel immune-based therapies and TB
vaccines are urgently needed to complement antitubercular drug
discovery [16-18]. We also believe that the immunotherapy is ought to be the indispensable part of therapeutic strategies against TB. A
large spectrum of diverse immunomodulators has been described for
treatment of TB and hepatitis . While some have shown clinical
utility, in most cases their mechanism is not well understood. This
drawback should be balanced against safety and therapeutic benefits
|Conversion of sputum smear from positive to negative
is considered as a critical endpoint of the efficacy of anti-TB
intervention. TB cases, especially those present with concomitant
HIV and HCV infections are extremely difficult to treat . Excessively
lengthy treatment duration and low success rates are common in
these patients. In the past we have conducted several clinical trials
of ATT in TB/HIV co-infected patients and based on this experience
we were not expecting sputum conversion rate better than 9-25%
after 6 months of continuous treatment. The present results reveal
that when V5 is used together with ATT it can enhance the effect
of TB drugs resulting in rapid sputum conversion in 94% of patients.
Such an outcome is clearly unprecedented and surpasses the effect of
immunomodulators we have tested in our prior studies [6-8]. Other
improvements indicative of beneficial outcome were drastic weight
gain and increase in hemoglobin levels. As a result 17 out 20 patients
(85%) were discharged from the hospital within one month.
|What is the rational explanation to the effect of V5? This
vaccine is derived from pooled blood of HCV+ and HBV+ donors.
It is well known that one-third of the world population harbor latent
Mycobacterium tuberculosis meaning that V5 preparation contains
TB bacterium present in donors’ blood. Therefore, the administration
of V5 could have triggered the immune response not only to hepatitis
viruses but also to tubercle bacilli. Whether this is true needs to be
verified by producing a vaccine candidate devoid of hepatitis viruses
but containing M. tuberculosis. To the best of our knowledge, the
only orally available, heat-killed therapeutic TB vaccine that has
shown promise in clinical studies is a preparation of Mycobacterium
vaccae (SRL172) discovered and developed by Stanford et al. .
Most TB vaccines that have recently entered clinical trials stage
are predominantly for prophylactic use and thus there is very little
information available in the literature regarding their potential in
treating TB [21-23].
|Placebo-controlled trial is thus needed to confirm our findings
and rule out non-specific factors that might have confounded the
effect of V5. Despite the small number of enrolled subjects and
short duration of the therapy the changes seen with every clinical
endpoint were highly significant. The time to clear-cut clinical benefit
appears to be only one month and, perhaps, even less as suggested
for example by fever normalization within few days. Considering that
conventional hepatitis and TB therapies require protracted treatment
periods, which can often last longer than one year, the observed
dynamics of response to the therapy is remarkable and gives us a
hope that in the future the duration of therapy can be shortened
substantially compared to current standards. Drug-resistant TB and
HIV co-infection are two major obstacles preventing the efficient
management of the disease . V5 can be an ideal tool against these
complications since it performs equally well compared to ordinary
TB and there were no significant differences in treatment duration
|In conclusion, our results indicate that one tablet of V5
administered daily can produce clinical effects seldom seen in prior
therapeutic vaccine trials. Hepatitis treatment options in developing
countries such as Ukraine are extremely limited due to the high cost
|of medications. A therapeutic vaccine derived from readily available
source certainly represents an affordable means to control chronic
HCV infection and TB at the same time. Additional studies are
warranted to develop better understanding of V5 properties and to
enlarge the current arsenal of safe and effective immunotherapies.
|We thank all volunteers who participated in this study. The wholehearted
support of clinicians, nurses and lab personnel who contributed their effort made
this study possible. We are grateful to our colleagues in Asia and Africa who shared
their experiences relating to the present study.
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