The effects of Avemar treatment on feline immunodeficiency virus infected cell cultures

Abstract Introduction In addition to standard highly active antiretroviral therapy protocols, complementary therapies using natural compounds are widely used by human immunodeficiency virus (HIV)‐infected human patients. One such compound is the fermented wheat germ extract (FWGE), named Avemar. Materials and methods In this study, we investigate the effects of Avemar in a feline‐acquired immunodeficiency syndrome model. MBM lymphoid cells were acutely infected by the American feline immunodeficiency virus (FIV)‐Petaluma (FIV‐Pet) and the European FIV Pisa‐M2 strains. FL‐4 lymphoid cells, continuously producing FIV‐Pet, served as a model for chronic infection. Crandell Rees feline kidney (CRFK) cells were infected by either FIV‐Pet or feline adenovirus (FeAdV) as a model for transactivation and opportunistic viral infection. Cell cultures were treated pre‐ and post‐infection with serial dilutions of spray‐dried FWGE (Avemar pulvis, AP), a standardized active ingredient in commercial Avemar products. Residual FIV and FeAdV infectivity was quantified. Results In a concentration‐dependent manner, AP inhibited replication of FIV strains in MBM and CRFK cells by 3–5 log. Low AP concentration prevented FIV‐Pet release from FL‐4 cells. Higher concentrations destroyed virus‐producing cells with cytopathic effects resembling apoptosis. AP strongly inhibited FeAdV production inside CRFK cells but not in HeLa cells. Adenovirus particles are then released via the disintegration of CRFK cells. Discussion This report is the first to describe the antiviral effects of Avemar. Further studies are required to confirm its in vitro and in vivo effects and to investigate the potential for its use as a nutraceutical in FIV‐infected felines or HIV‐infected humans. Conclusion Avemar, as a single nutraceutical, inhibits FIV replication and destroys retrovirus carrier cells. An important conclusion is that prolonged Avemar treatment might reduce the number of retrovirus‐producing cells in the host.


INTRODUCTION
The immune system's response to human immunodeficiency virus (HIV) infection leads to metabolic changes and weight loss with significant morbidity and mortality. Highly active antiretroviral therapy (HAART) has significantly modified the natural course of acquired immunodeficiency syndrome (AIDS), transforming it into a chronic disease; however, cells carrying integrated HIV cannot be eradicated by HAART, and a small fraction of the virus escapes from control.
So far, the search for drugs capable of selectively eradicating HIV from humans or feline immunodeficiency virus (FIV) from cats has failed.
HIV-infected patients frequently use complementary and alternative medicine therapies to improve nutritional status, treatment outcomes and alleviate the side effects of antiretroviral therapy. A proprietary fermented wheat germ extract (FWGE), known as Avemar, is a widely used immunomodulatory and anticancer nutraceutical (Boros et al., 2005). Avemar has been also used in the veterinary practice under different formulations, Immunovet for farm animals and pets or, for horses, eCalm (Stipkovits et al., 2004, Sutton et al., 2018. It is produced under good manufacturing practice conditions . Avemar pulvis (AP, FWGE powder), the active ingredient, is standardized to methoxysubstituted benzoquinone marker compounds, that is 2,6-dimethoxyp-benzoquinone (2,6-DMBQ) and 2-methoxybenzoquinone (2-MBQ).
These compounds are liberated as aglycones by the action of glycosidases of the fermenting organism, Saccharomyces cerevisiae. AP contains all the water-soluble materials of fermented wheat germ liquid, encapsulated with maltodextrin and spray-dried. A single dose (17 g) of the commercial Avemar granulate contains 8.5 g AP plus added flavouring ingredients. FWGE is a complex mixture of thousands of molecules. Most of them have not been chemically identified yet (Telekes et al., 2009). Avemar has a direct pro-apoptotic effect in lymphoma cells and boosts host immune effector mechanisms (Barisone et al., 2018). It initiates intrinsic mitochondrial-dependent apoptotic signalling in tumours (Bencze et al., 2020), restores normal immune functions in immunocompromised animals , reverses the transformed metabolic phenotype (Boros et al., 2005), inhibits ribonucleotide reductase (Saiko et al., 2007), cleavages poly(ADP)ribose polymerase (PARP) (Comin-Anduix et al., 2002) and shows robust antiproliferative effects and triggers tumour cell death through apoptosis (Zhurakivska et al., 2018). AP has a firmly established safety profile (Heimbach et al., 2007) and an independent panel of medical, food safety and toxicology experts confirmed Avemar's generally recognized as safe status in accordance with US FDA's regulations (Hoffman et al., 2005). In countries of the European Union, Avemar has been approved as a dietary food for cancer patients and has since been marketed as a non-prescription medical nutriment in other parts of the world (Patel, 2014). In cancer and autoimmune clinical studies, Avemar has proven to be advantageous in terms of disease progression, overall survival and clinical outcome (Mueller & Voigt, 2011).
FIV-infected cats can serve as informative animal models for AIDS studies (Matteucci et al., 1995). FIV is a widespread pathogen of domestic cats (Pedersen, 1993). Feline isolates are classified into subtypes A (USA-California and Europe), B (Europe, Japan, and USA except for California), C (Canada) and D (Japan) (Hohdatsu et al., 1996). So far, FIV infection has been detected in 37 felid species living free or in zoological collections (O'Brien et al., 2012). FIV-infected cats are susceptible to opportunistic infections and cancer development, thus offering a strong parallel to related virus-based pathologies in humans.
Consequently, viral interactions, both related to AIDS and to agerelated complications, can be studied in the feline AIDS model (Ongrádi et al., 2013).
Adenoviruses (AdVs) are widespread among mammals and can cause severe infections. In human AIDS, early genes (E1A, E1B) of AdVs have been shown to transactivate HIV and consequently promote AIDS progression (Kliewer et al., 1989). AdVs, reactivated from latency, elicit opportunistic infections. Before the introduction of HAART, approximately 20% of AIDS patients died of untreatable gastroenteritis. Species C AdV types are the more frequent causative agents, but unusual B and D intratypic recombinants are also produced and excreted in the stool and urine of AIDS patients (Hierholzer, 1992).
After bone marrow or hematopoietic stem cell transplantation, reactivated AdVs can elicit prompt lethal complications or lifelong sequelae (Lion, 2019). Inhibition of AdV expression might provide clinical benefits to AIDS patients. AdV infections have not been widely studied in cats and Felidae. We first described European and partially American AdV epidemiology and characterized the first feline adenovirus (FeAdV) isolate. It is related to but distinct from human adenovirus 1 (Ongrádi et al., 2019).
In the present paper, our studies on Avemar in the feline AIDS model are presented.

Avemar pulvis
AP and Avemar granulate were obtained from the manufacturer (Biropharma Kft., Kunfehértó, Hungary). The samples were freshly dissolved in sterile phosphate-buffered saline at 10 mg/mL concentration, cleared by centrifugation, filtered through a 22 µm filter and stored in the freezer at −20 • C. Further dilutions were prepared in complete Dulbecco's modified Eagle's medium (DMEM) or in complete RPMI-1640 medium, depending on the culture conditions of the particular cell line studied.

FIV stocks and virus titration
A stock solution of FIV-M2 was obtained by infecting MBM cells with FIV-M2. The supernatant fluid of FL-4 cells was used as a source of FIV-Pet. On day 10, viruses were harvested in the supernatant fluid by centrifugation and stored as 1 mL aliquots at −80 • C. Samples were titrated on CRFK cells as described (Yamamoto et al, 1991, Matteucci et al, 1995. Median tissue culture infective dose (TCID 50 ) values were determined by the Reed-Muench method (Reed & Muench, 1938), FIV-Pet titred at 10 5 syncytium forming unit/mL (SFU/mL), whereas FIV-M2 did so at 10 4 SFU/mL. FIV production was quantitated by p24 Diego, CA, USA) as previously described (Ongrádi et al., 2019). The final concentration of the virus was 1.35 × 10 7 infectious unit/mL (IU/mL).

Monitoring the effect of Avemar pulvis on cell cultures
Cell growth, CPE and viability determined by trypan blue exclusion were monitored daily by light microscopy. Cytotoxicity of AP, as determined by the growth arrest of the cells in the logarithmic phase, was determined by EZ4U, a fourth-generation, nonradioactive cell proliferation and cytotoxicity assay (Biomedica Medizinprodukte GmbH, Wien, Austria). MBM, FL-4, CRFK and HeLa cells were aliquoted with serial AP dilutions at final concentrations between 0 and 5000 µg/mL. Samples containing 1 × 10 4 cells were collected on days 1, 2, 3 and 7 and complemented with 20 µL substrate (tetrazolium). Optical density was read as above. The ratio of living cells was calculated relative to untreated controls (%).

Statistical analysis
All experiments were carried out in triplicate or quadruplicate. The data were expressed as the mean ± standard error of mean. Statistically significant differences were evaluated by a two-sample t test (p < 0.05 was deemed to be significant).

Cell growth arrest and cytotoxicity elicited by Avemar pulvis
The effects of serial dilutions of AP on the replication, viability and morphology of cells were monitored daily. AP of 5000 µg/mL exerted strong cytotoxicity on all cell types (data not shown). MBM cells were stimulated by low AP concentrations (up to 1000 µg/mL) up to day 7, but from this concentration onwards, a sharp onset of cytotoxicity was demonstrated ( Figure 1A). A smaller fraction (13%-17%) of cells did not grow but remained viable as demonstrated by trypan blue (data not shown). During the first days of culture, the replication of FL-4-Pet cells ( Figure 1B Aliquots of cells were removed on days 1, 2, 3 and 7, and the ratio of living cells was determined by optical density. *p < 0.05, **p < 0.001.

Effect of Avemar pulvis on FIV-infected CRFK cells
AP concentrations of 250-1000 µg/mL transiently delayed the onset and progression of CPE in a dose-and time-dependent manner (data not shown). Virus production was quantified in supernatants F I G U R E 4 Quantitation of the residual feline immunodeficiency virus-Petaluma (FIV-Pet) infectivity in Avemar-treated FL-4 cultures. Samples from supernatant fluids and disrupted cells were taken on day 4 after Avemar treatment. Residual infectivity was titrated on Crandell Rees feline kidney (CRFK) cells. *p < 0.05, **p < 0.001.

F I G U R E 5
The effect of Avemar on feline immunodeficiency virus-Petaluma (FIV-Pet) p24 antigen production by Crandell Rees feline kidney (CRFK) cells. Antigen concentration in the supernatants fluids was determined according to the standards of the kit. *p < 0.05, **p < 0.001. harvested at 0, 5 and 9 days pi by measurement of p24 content. The effect of representative low and high AP treatments is shown in   Figure 6A,B). In contrast, 24 h AP pretreatment very strongly inhibited hexon antigen production of high input virus (10 moi) infected cultures in a dose-dependent manner. Post-infection AP treatment also exerted a significant inhibitory effect, though, to a lesser extent ( Figure 6B). FeAdV replication following low input (moi 1 and 0.1) was very strongly inhibited by increasing AP concentrations when cells had been 24 h pretreated with the drug (Figure 7A,B).

Effect of Avemar pulvis on FeAdV replication in HeLa and CRFK cells
FeAdV hexon antigen production by HeLa cells was hardly influenced by AP pre-or post-treatment using low or high input virus ( Figure 6C). Infection of MBM cells was used to model acute FIV-M2 and FIV-Pet infection, the former having been previously shown to replicate at higher titres (Matteucci, et al., 1995, Tarcsai et al., 2021. A single dose of Avemar strongly inhibited the CPE effect of acute FIV infection with a simultaneous reduction in the viral load of the media. FIV-M2 appeared to be slightly more sensitive to the antiviral activity of AP than FIV-Pet. Earlier studies have shown that FIV-M2-infected MBM cells lysed without syncytia formation (Matteucci et al., 1995), and we have previously shown that acute FIV-M2 infection of MBM cells elicits programmed cell death (Tarcsai et al., 2021). FIV is known to induce apoptosis of acutely infected, IL-2-dependent T-lymphoid MYA-1 cells (Ohno et al., 1994) and peripheral blood lymphocytes of cats (Bishop et al., 1993). or phytic acid, phytate) has been identified as an essential cofactor of this budding process, namely the IP6-driven HIV-1 viral protein (Gag) assembly has been shown a mandatory mechanistic part of HIV-1 virion budding (Pak et al., 2022). IP6 is widespread in cereal grains (Hidvégi & Lásztity, 2002). Wheat germ, which is the raw material of Avemar manufacturing, may contain up to 3.9% of IP6 (Schlemmer et al., 2009). Fermentation has been shown to degrade the hexa form of phytic acid by increasing phytase activity, resulting in the generation of lower myo-inositol polyphosphates (IP5, IP4, IP3, IP2, IP1) and also myo-inositol (Kumar & Anand, 2021). Consequently, Avemar, the fermentation product of wheat germ, contains no IP6, only its fragments (unpublished data). Although the exact mechanism is still to be explored, we assume that phytate fragments in AP may be factors in the anti-budding mechanism of Avemar via allosteric inhibition of Gag assembly. Thus, the cofactor role of IP6 in HIV morphogenesis may be impeded by AP. Further support of this proposed mechanism regarding the inhibition of HIV budding came from a study in which a synthetic myo-inositol pentakisphosphate derivative (L-HIPPO) was shown to intercept Gag protein assembly, thus inhibiting HIV-1 virion budding. This mechanism of HIV eradication in reservoir cells has been named 'lock-in and apoptosis' , because the 'locked-in' HIV induces apoptosis of the host cells (Tateishi et al., 2017).
Avemar treatment induces cellular death in both acute and chronically infected cells, and the associated changes in cell morphology suggest that this could be due to apoptosis. A previous attempt to induce apoptosis of FL-4 cells was unsuccessful (Kakizaki et al., 2006). Alpha-amanitin inhibited cell proliferation and FIV-Pet production by FL-4 cells (Tanabe et al., 2021) and also inhibited adenovirus replication (Ledinko, 1971), but, due to its toxicity in vivo, clinical application is impossible.
In  -Boja et al., 2002). In HeLa cells, hexon antigen production and virus release were not restricted by AP. HeLa cells harbour integrated human papillomavirus (HPV) genes. It has been shown that E6 and E7 gene products of HPV inhibit apoptosis (Tan et al., 2012) and transactivate adenovirus (Howley et al., 1989)  This has to be explored by future studies.

CONCLUSIONS
In summary, we can conclude that Avemar, as a Cells chronically producing retroviruses evade apoptosis. One of the most important observations in our study is that Avemar interrupts the replication of such cells. It may thus cautiously be supposed that prolonged Avemar treatment will gradually reduce and finally eradicate these retrovirus-producing cells in the host. If we suppose that AP inhibits FIV and FeAdV, we may also suppose that this nutraceutical may also inhibit HIV and human AdVs. Thus, these aspects ought to be explored in future studies. The latter should include clinical trials with HIV-infected humans receiving HAART and adjunctive Avemar, as well as clinical studies with FIV-infected pets and endangered large cats.