Anelloviruses versus human immunity: how do we control these viruses?

Abstract One continuous companion and one of the major players in the human blood virome are members of the Anelloviridae family. Anelloviruses are probably found in all humans, infection occurs early in life and the composition (anellome) is thought to remain stable and personal during adulthood. The stable anellome implies a great balance between the host immune system and the virus. However, the lack of a robust culturing system hampers direct investigation of interactions between virus and host cells. Other techniques, however, including next generation sequencing, AnelloScan-antibody tests, evolution selection pressure analysis, and virus protein structures, do provide new insights into the interactions between anelloviruses and the host immune system. This review aims at providing an overview of the current knowledge on the immune mechanisms acting on anelloviruses and the countering viral mechanisms allowing immune evasion.


Introduction
Making up over 70% of the healthy human blood virome, anelloviruses are considered to be the principal constituent of the human virome (Freer et al. 2018, Liou et al. 2022 ).Dominating not only within an individual but also worldwide, they are hypothesized to be present in the entire human population (Niel et al. 2000, Virgin et al. 2009 ).To date, four human infecting genera have been discov er ed: Alphator quevirus (Torque teno virus; TTV), Betatorquevirus (Torque teno mini virus; TTMV), Gammatorquevirus (Torque teno midi virus; TTMDV), and the r ecentl y discov er ed fourth Hetorquevirus (Torque teno hominid virus) (Varsani et al. 2021 ).Information on Hetorqueviruses remains to be explored.The composition of anelloviruses infecting an individual is referred to as the anellome (Kaczorowska and van der Hoek 2020 ).The composition of such anellome is highly individualized and differs not only in the genera present but also in the corresponding concentrations of anelloviruses (Kaczorowska et al. 2022b ).After its initial establishment, which can be traced back to the first months of life, the anellome differs in its composition when compared to that of adults.A young infant's anellome primarily harbors TTMVs and TTMDVs soon follo w ed b y the TTVs (Kaczorowska et al. 2022a ).It is thought that only over time the anellome is shaped into its adult version, where the anellome is often dominated by TTVs (Kaczorowska et al. 2022b ), and richer in men compared to women (Cebriá-Mendoza et al. 2023 ).As research on the period between the second year of life toward adulthood has not been conducted, the transition process is yet to be elucidated.Studies on the anellome of adults r e v ealed that the composition in healthy people remains relatively stable over decades (Maggi et al. 2003, Kaczorowska et al. 2022b ).Information on the dynamics or de v elopment of the anellome in elderly is not a vailable , although it is known that anelloviruses increase with age (Cebriá-Mendoza et al. 2021 ).Despite incr easing r esearc h efforts, the scientific community has been unable to relate anelloviruses to a disease, implying anelloviruses could be a commensal component of the human virome (Kaczorowska and van der Hoek 2020 ).
With its genome size ranging from only 2.0 to 3.9 kb, anelloviruses are small, single stranded, and circular DNA viruses of negative sense, that contain ov erla pping open r eading fr ames (ORF) and an untranslated region (Miyata et al. 1999, Jones et al. 2005, Ninomiya et al. 2007 ).The protein encoded by the ORF1 gene is considered to be the greatest in size and encodes the viral capsid (Qiu et al. 2005, Sarker et al. 2016 ).The protein encoded by ORF2 has, in addition to being essential for virion assembly (Nawandar et al. 2022 ), a potential regulatory function, suggested to interfere with immune responses of the host (Zheng et al. 2008 ).TTV-deriv ed a poptosis-inducing pr otein (TAIP), encoded within the same region as ORF2 but different reading frame, is thought to play a role in triggering apoptosis of the host cell (Kooistra et al. 2004, Hino and Prasetyo 2009, Prasetyo et al. 2009 ).The function of the proteins expressed by ORF3 and spliced mRNAs ORF1/1, ORF1/2, ORF2/2, and ORF2/3 are yet to be deciphered.
The lack of a functioning culturing method, a system with infectious anellovirus particles that can be passa ged, (r e vie wed elsewher e Kaczor owska and v an der Hoek 2020 ), has limited r esearc h on anello viruses .Ne v ertheless, using clinical samples with v arying immune statuses enables researchers to investigate the relationship anelloviruses may have with the human immune system.It is known that anelloviruses are susceptible to interferon (IFN).In vitro IFN-γ treatment of a TTV-transfected Hodgkin lymphoma cell line L428 and a TTV-transfected kidney cell line 293 r e v ealed inhibited TTV DNA replication (de Villiers et al. 2009 ).In addition, people treated with IFN-α-due to an hepatitis C virus infection-gener all y sho w ed decr easing TTV concentr ations in blood (Chay ama 1999, To y oda et al. 1999, Nishizaw a et al. 2000, Maggi et al. 2001 ).Whether this vulnerability to IFN is also applicable to TTMV and TTMDVs is thus far unknown.Next to that, se v er al studies have investigated TTVs, or in some studies total anello viruses , in or gan imm unosuppr essed tr ansplant r ecipients and found an inv erse corr elation between vir al load and immune activity (Burra et al. 2008, Liu et al. 2021 ).In line with the abovementioned findings are the results presented by Shibayama et al. ( 2001 ) who r e v ealed that individuals with AIDS display high pr e v alence and high concentrations of TTVs (Shibayama et al. 2001 ).Even though these findings show no causal relationships, and thus do not provide insights into specific mechanisms of virus-host interactions, these findings do indicate that the anellome must be under (some) control by immune surveillance.
In this r e vie w, we pr esent an ov ervie w of the mec hanisms discov er ed so far that that are likely involved in immune surveillance, as well as the mechanisms applied by anelloviruses to esca pe imm une surv eillance.

APOBEC3 editing
One element of the innate imm une defense, a polipopr otein B mRNA-editing catal ytic pol ype ptide-lik e 3 (APOBEC3 or A3) exhibits antivir al pr operties due to the ability to deaminate cytosine (C) to uracil (U) in the viral genome (reviewed by Pecori et al. 2022 ).The introduction of mutations is thought to result in the production of dead-end genomes and thus impaired viral replication (Ball et al. 1999, Tsuge et al. 2010 ).The APOBEC3 family consists of se v en pr oteins: A3A, A3B, A3C, A3D, A3F, A3G, and A3H, that differ based on their intracellular location, and the nucleotide context they prefer to deaminate (Pecori et al. 2022 ).APOBEC3mediated editing has been found in the genome of many DNA and RNA viruses, such as herpesviruses , parvo viruses , hepatitis B virus, Human Immunodeficiency Virus (HIV), human papilloma viruses , and corona viruses (Noguchi et al. 2007, Vartanian et al. 2008, Kukimoto et al. 2015, Warren et al. 2015, Ratcliff and Simmonds 2021, Kim et al. 2022 ).
First evidence for interactions between anelloviruses and APOBEC3 has already been suggested in 1999 by Ball et al. ( 1999 ), who found APOBEC3 editing in 1 out of 93 TTV reads using Sanger sequencing.These findings were consolidated in 2010 by Tsuge et al. ( 2010 ), who performed a study, also using Sanger sequencing, demonstrating that APOBEC3 editing could indeed be found in TTV genomes (Tsuge et al. 2010 ).Based on the specific nucleotide contexts in which the mutations were found, A3G and A3F were suggested as responsible candidates.
While Ball et al. ( 1999 ) and Tsuge et al. ( 2010) looked specifically at TTVs, a study of Poulain et al. ( 2020 ) predicted that APOBEC3 is most pr obabl y also acting on TTMVs and TTMDVs (Poulain et al. 2020 ), as A3-recognition motif depletion was found for all thr ee gener a.These pr edictions wer e r ecentl y confirmed by us.By next generation sequencing, we investigated hypermutations in 64 anellovirus isolates in healthy people, and r e v ealed that all three human anellovirus genera are affected by C to U deamination, with some isolates more vulnerable to deamination than others (Timmerman et al. 2022 ).The U-containing genomes were pac ka ged inside intact virus particles, which w e kno w because the clinical materials wer e tr eated with DNAse prior to analysis.The fact that uracils were found in the genomes within intact virus particles suggests that editing m ust hav e occurr ed at the late stage of the replication cycle, since the mRNA encoding an intact viral capsid could not have been transcribed from edited DN A. Accor ding to the observed nucleotide context, we considered A3C, A3D, A3F, and A3H as potential candidates acting on the negative sense anellovirus genome (Timmerman et al. 2022 ).Of note, APOBEC3 activity did not seem to change an anellome, as isolates that were most affected by editing wer e r ar el y lost in their hosts during the > 30 years that we were able to follow c hr onic anellovirus infections in individuals.
The mutational activity of the APOBEC3 protein can be enhanced by IFN-α and IFN-γ (Peng et al. 2006, 2007, Noguchi et al. 2007, Koning et al. 2011 ).Inter estingl y, in the experiments performed by Tsuge et al. ( 2010 ) alanine aminotr ansfer ase flar e ups found in hepatitis B patients, associated with increased IFN-γ levels, led to APOBEC3 editing in the hepatitis B virus, and-at the same time-TTV le v els dr opped below their detection limit (Tsuge et al. 2010 ).This suggests that the decrease in TTV le v els, occurring during IFN-γ and IFN-α treatment, may potentially be due to APOBEC3 editing activation.

TLR-9 activ a tion
Another unknown is Toll-Like receptor-9's (TLR-9) involvement in anellovirus recognition.TLR-9 is a Toll lik e rece ptor present mainl y intr acellularl y in antigen pr esenting cells, r ecognizing unmethylated DNA.Although TLR9 triggering was found after exposure to TTV-DNA in mouse spleen cells (Rocchi et al. 2009 ), no studies have thus far confirmed TLR-9 involvement using whole viruses , e .g. anello viruses obtained from clinical samples like blood or saliva.

Antibody response
Antibodies, as part of the ada ptiv e imm une system, ar e an important immune defense mechanism against viral infections (Delves and Roitt 2000 ).They may, ther efor e, also play a crucial role in k ee ping anelloviruses in c hec k.Indeed, antibodies a gainst anello viruses ha ve been detected.In 2000, Itoh et al . ( 2000 ) detected that purified virions were generally immune complexed with IgG.Next, Ott et al. ( 2000 ) expressed an almost full length TTV genotype 1 ORF1 in Esc heric hia coli and performed a serological study in 70 individuals, including blood donors, hepatitis patients, and c hildr en.They found that 98.6% of the individuals wer e positiv e for antibodies a gainst this pr otein, wher eas onl y 76% of the individuals were PCR positive for TTV.In other studies a m uc h lo w er pr e v alence of antibody r eactivity was found.Tsuda et al. ( 1999 ) compared antibody prevalence in TTV genotype 1 PCR positive and negative individuals by testing reactivity to whole virions (Tsuda et al. 1999 ).Virions (10 5 TTV DNA copies per ml) wer e obtained fr om a feces suspension, and imm une pr ecipitation found antibodies in 17% of the healthy individuals PCR positive for TTV, whereas 29% of the healthy individuals PCR negative for TTV show antibodies against the virus .T his link between PCR-negativity and increased frequency of finding ORF1 antibodies w as, ho w e v er, not found in two other studies.For TTV genotype 1 (Handa et al. 2000 ) as well as for genotype 6 (Kakkola et al. 2008 ), IgG antibodies in serum were found at higher frequency in individuals that were PCR positive for TTV.It is noteworthy though that the TTV-PCRs used in the various studies can differ, either by being broad and detecting a large variety of TTV isolates, or narrow with detecting only one (TTV genotype 1) isolate.PCR-detection of a few or many TTV isolates in a sample may explain the discrepancies in the abovementioned studies.Mor eov er, anellovirus pr oteins ar e extr emel y div erse, species and gener a shar e at most 69% and 44% of its ORF1 coding r egion, r espectiv el y (Varsani et al. 2021 ).Individuals can carry a wide variety of infecting anelloviruses (Kaczorowska et al. 2022b ), and antibody cross-reactivity may or may be limited between species, genotypes or genera.
As antibody-mediated neutralization of a virus depends on the epitopes being tar geted, unv eiling the locations where antibodies bind is of interest.In 2022, Venkataraman et al. ( 2022 ) used AnelloScan, a pha ge imm unopr ecipitation-sequencing assay (PhIp-Seq) to analyze the binding ability and affinity of antibodies to TTV, as well as TTMV-and TTMDV-derived linear peptides from ORF1, ORF2 ORF3, and TAIP (Venkataraman et al. 2022 ).The assay was not limited to a single genotype of each genus, as no less than 800 different human anelloviruses isolates (326 TTVs, 357 TTMVs, and 146 TTMDVs) were tested in the AnelloScan.Their results indicate that the protein encoded by the ORF1 gene is the most common target of antibodies, follo w ed b y ORF2, whereas reactivity to ORF3 or TAIP was r ar el y found (Venkatar aman et al. 2022 ).Ho w e v er, one m ust k ee p in mind that the 3D conformation of the phage-displayed peptide library might differ from the conformational shape of the full protein used in other studies.Kakkola et al. ( 2008 ) expressed six ORFs of TTV genotype 6 in ele v en differ ent combinations in bacterial cultur es and insect cells as arginine-depleted constructs (Kakkola et al. 2008 ).They found, in contrast with the findings of Venkataraman et al. ( 2022 ), highest IgG r eactivity a gainst the pr otein encoded by ORF2 (28.6%) and ORF2/3 (23.8%), follo w ed b y ORF1 (14.3%),ORF2/2 (5.3%), and ORF1/1 (5.6%).No reactivity against ORF1/2 could be observed.The response to w ar ds ORF1 w as strongest for the C-terminal part of the protein, and response against the N-terminus was lacking (Kakkola et al. 2008 ).The immunodominance of the C-terminal part of ORF1 was also found in the AnelloScan (Venkataraman et al. 2022 ) .
The presence of antibodies targeting anelloviruses prompted a deeper investigation into the potential implications for anellovirus survival over time.Kakkola et al. ( 2008 ) were able to investigate antibody response of four individuals for se v er al years (Kakkola et al. 2008 ).One of the individuals remained PCR positive for TTV genotype 6 for over 6 years, and was genotype 6-ORF1 IgG positiv e thr oughout the whole follow-up.A second individual became PCR-positive for genotype 6 during follow up, yet did not develop antibodies to the genotype 6-ORF1.Instead antibodies recognizing genotype 6-ORF2 protein were found.Interestingly, following this ORF2 antibody ser oconv ersion, this individual became PCR-negative for genotype 6, and remained negative for 5 years until the end of the follow up.Venkataraman et al. ( 2022 ) also inv estigated antibodies tar geting ne wl y intr oduced anelloviruses in people receiving blood transfusions (Venkataraman et al. 2022 ).They found that antibodies specifically recognizing the newly introduced viruses were produced in only a minority of the recipients.Ho w e v er, 5 months post transfusion > 70% of the ne wl y introduced isolates, whether with or without produced reactive antibodies, did not stabilize themselves in the anellome, suggesting that other immune pressure , e .g. T-cell immunity (see below), may also play a role in eradication of newly introduced anello viruses .

T-cell immunity
To date, no studies investigating T-cell immunity and anellovirus dynamics have been reported, ho w ever, b y looking at immunodeficiencies with low CD4 + T cell counts some indir ect e vidence on T-cell-mediated control may be obtained.Shibayama et al. ( 2001 ) and Liu et al. ( 2021 ), looked at anelloviruses in treatment naïve people with HIV-1 (PWH) (Shibayama et al. 2001, Liu et al. 2021 ).Through comparison of the viral loads versus the CD4 + T cell count in PWH using PCRs and next-generation sequencing, they found an inverse correlation; individuals that had a lo w er CD4 + T cell count also displayed higher pr e v alence and higher concentrations of TTVs.Although these results do not provide insights into specific mechanisms, the results do indicate that the anellome must, at least in part, be controlled by T-cell immunity.
Another indirect T-cell study was done by Sospedra et al. ( 2005 ).They isolated and expanded CD4 + T cells from cerebrospinal fluid of a MS patient, and predicted that these cells recognize peptide motifs from the arginine-rich region of the TTV ORF1 protein (Sospedra et al. 2005 ).It must be mentioned though that arginineric h motifs ar e found in v arious viruses and autoantigens, and the r ecognition, ther efor e, does not necessaril y pr edict dir ect Tcell r eactivity a gainst anelloviruses specificall y.Additionall y, the peptides wer e r ecognized at moder ate concentr ations, whic h indicates low avidity of the T-cells, pr obabl y too low to be able to clear TTVs (Sospedra et al. 2005 ).
People who r eceiv e allogenic organ transplants are treated with imm une suppr essiv e medication, e.g.corticoster oids and/or anti-T-cell induction imm unosuppr essants.Focosi et al. ( 2015 ) sho w ed decreased TTV load in kidney or liver transplant patients 7 days after anti-T-cell drug administration, by either administration of anti-thymocyte globulin or basiliximab (Focosi et al. 2015 ).This TTV dr op also corr elated with the r eduction of number of l ymphocytes in the first 15 da ys .Inter estingl y, the concentr ation of anelloviruses sho w ed a 2-fold increase 30 days after transplantation, while the lymphocyte concentration remained low.Other medications known for T-cell inhibition are Calcineurin inhibitors (tacr olim us/cyclosporine) (Laupacis et al. 1982 , Clipstone andCr abtr ee 1992 ).In liv er tr ansplant r ecipients, a rise in TTVs is observed 3 months after transplantation, and this rise in TTV concentrations is higher with increasing concentrations of calcineurin inhibitors (Focosi et al. 2014 ), indicating that T-cells play a role in the control of anello viruses .Ho w ever, so far no study has specificall y searc hed for T-cells that become activated upon r ecognition of distinctiv e anello virus antigens .Needless to sa y that further r esearc h is necessary to acquir e (mor e) understanding of anellovirus imm une surv eillance via T-cells, but the large div ersity in eac h individual anellome may hamper easy measuring of anellovirus-specific T-cell reactivity.An AnelloScan-like appr oac h (with > 800 isolates cov er ed) may also be needed here.

Antibody immune e v asion
Anello viruses ma y escape host B-cell-mediated immunity by evolving at an antibody recognition site, to such an extent that an antibody can no longer bind (Liou et al. 2022 ).One potential site of ORF1 where this process may occur is the hypervariable P2 region, part of the spike domain that forms a crown-like structure in the capsid of anelloviruses (Liou et al. 2022, Butkovic et al. 2023 ).Nishizawa et al. ( 1999 ) investigated sequence divergence by sequencing TTV genotype 1 in sera of two patients over time (Nishiza wa et al. 1999 ).T hey found that the majority of the amino acid changes occurred in this hypervariable P2 region.In addition, we hav e r ecentl y inv estigated the e volution of six individual anellovirus isolates, intr ahost, ov er the span of 30 years and confirmed that the majority of ORF1 nucleotide substitutions that lead to a change of an amino acid (positive selection) occur in this hyperv ariable r egion (Kaczor owska et al. 2023 ).It is most likely that the strong pressure to mutate in the hypervariable P2 region in ORF1 is related to the benefit these amino acid changes provide in escaping antibody recognition.Kakkola et al. ( 2008 ), also found in one individual-follo w ed over 6 years-that presence of antibodies recognizing genotype 6-ORF1 did not lead to clearance of the genotype 6 TTV (Kakkola et al. 2008 ).Although not tested, as no longitudinal genotype 6 sequencing was done, persistence in this individual may have been due to antibody escaping mutations in the hypervariable region.

Suppression of NF-κB
A k e y pathway factor in innate and ada ptiv e imm unity is NF-κB (nuclear factor kappa light chain enhancer of activated B cells) signaling (Oeckinghaus and Ghosh 2009 ).By activation, NF-κB signaling regulates gene expression of various proinflammatory genes including inflammasome activation, and activation of innate immune cells .T he gene family codes for fiv e pr oteins: P50, P52, P65, RelB, and C-Rel (Hayden and Ghosh 2012 ).With respect to anello viruses , Zheng et al. ( 2008 ) r e v ealed, b y using a w estern blot, that the protein encoded by ORF2 is able to suppress the proinflammatory NF-κB pathw ay b y do wnr egulating the tr anslocation of the proteins p50 and p65 into the nucleus through inhibition of Inhibitors-κB α (I κB α) degradation (Zheng et al. 2008 ).Normally, the binding of I κB α to NF-κB in the cytosol pr e v ents the nuclear translocation of NF-κB.Only after the phosphorylation of I κB α by I κB α kinases (IKK), leading to degradation of I κB, releases NF-κB, whic h allows tr anslocation into the nucleus, and results in transcription of genes of proinflammatory genes (Oeckinghaus and Ghosh 2009 ).Ther efor e, by the ORF2 mediated inhibition of IKK, NF-κB translocation may be prevented, which subsequently inhibits the production of inflammatory responses threatening anellovirus survival (Zheng et al. 2008 ).As NF-κB can also function as a transcription factor of certain proinflammatory genes, ORF2 protein can, thus also indirectly cause downregulation of certain proinflammatory cytokines transcription, which was indeed seen for interleukin-6 (IL-6), interleukin-8 (IL-8), and COX-2 (Zheng et al. 2008 ).

Anello virus microRN A
MicroRN A (miRN As) are small single stranded, noncoding RNAs, which can be encoded by the host, but also by the virus (Kincaid and Sulliv an 2012 ).Vir al miRNAs can gener all y play a role in the extension of the lifespan of infected cells, e v asion of the immune system, and the regulation of lytic infection.Anelloviruses have been found to express both single-stranded and double-stranded miRNAs, of which at least one transcript was found in plasma exosomes, in as m uc h as 98 out of 102 subjects (Vignolini et al. 2016 ).Ho w e v er, mor e r esearc h is needed to elucidate the potential immune modulating effects of anellovirus encoded miRNAs.

Concluding remarks
Ever since their discovery in 1997 (Nishizawa et al. 1997 ), r esearc hers hav e tried to shed light onto the interactions anello viruses ha ve with their human host.One main question remains, ho w e v er, unanswer ed: How can viruses that are so omnipresent, with continuous virus presence in the circulation, be tolerated by their host?Other DNA viruses known for their c hr onic infection and lifelong presence in humans (e.g.varicella zoster virus , parvo viruses , and herpesviruses) turn to latency after acute infection, and only during reactivation these viruses can be detected in blood or other sites of the body.Anelloviruses behave differ entl y with their continuous presence in blood and no clear latency .In theory , it could be that tolerance due to anellovirusspecific immune exhaustion plays a role.Immune tolerance is for instance seen in c hr onic infection of the liver with the hepatitis B virus, where high hepatitis B DNA and surface antigen (HBsAg) le v els in blood results in tolerance due to (T-cell) immune exhaustion (Fisicaro et al. 2020 ).
Anelloviruses show a remarkable variability, probably caused by thousands or e v en millions of years of evolution.Not only does this v ariation pr e v ent the de v elopment of simple immunological assays like antibody or antigen ELISA, the high diversity complicates a study on anellovirus-host interactions even further.We suggest that in futur e r esearc h, anelloviruses should not be a ppr oac hed as one virus, but se v er al. Differ ences between subgenera and/or isolates were detected regarding increased anellovirus load in immunosuppressed individuals (Blatter et al. 2018 ), APOBEC3 susceptibility (Timmerman et al. 2022 ) and antibod y reacti vity (Venkataraman et al. 2022 ).It would be of inter est to inv estigate whether subgener a, or isolates, ar e mor e imm unogenic or toler ogenic compar ed to others.When doing suc h r esearc h it may e v en be needed to take the (genetic) makeup of the infected person into account, which might also dictate the make-up of someone's anellome.
Together, the interactions between anelloviruses and the host immune system form an intricate balance that results in the persistence of an anellome in its host.Research is strongly limited by the lack of an adequate culturing system, or animal model and it forces r esearc hers to use indir ect measur es to study virushost interactions .T here is a reasonable chance that, apart from hepatocytes , immune cells , including granulocytes , or stem cells may actually facilitate replication of the virus (Itoh et al. 2000, Luo et al. 2000, Okamoto et al. 2000, Yu et al. 2002, Hu et al. 2005, Kosulin et al. 2018 ; r e vie w ed b y Kaczoro wska and van der Hoek 2020 ; r e vie w ed b y Taylor et al. 2022 ).Lymphoc ytes, although pr obabl y not the activated T-cells (Li et al. 2015 ), could also be susceptible for anellovirus infection (Yu et al. 2002, Leppik et al. 2007 ).Immune cells having a double role in anellovirus persistence-combatting the virus, and at the same time facilitating replication of the virus-may complicate studies on anellovirus-imm une inter actions e v en mor e.