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Herpes Simplex Virus Infection, Acyclovir and IVIG Treatment All Independently Cause Gut
1
Dysbiosis.
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3
Chandran Ramakrishna
1
, Stacee Mendonca
1,
Paul M. Ruegger
2
, Jane Hannah Kim
2
, James
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Borneman
2
and Edouard Cantin
1*
.
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6
Department of Molecular Immunology
1
, Beckman Research Institute of City of Hope, Duarte, CA
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91010 and Department of Microbiology and Plant Pathology
2
, University of California, Riverside,
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CA 92521.
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10
*Corresponding Authors: Edouard M. Cantin
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Beckman Research Institute of City of Hope
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Department of Molecular Immunology
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Fox Plaza North, Room 100B
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1500 E. Duarte Rd, Duarte CA 91010-3012
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Phone: +1 (626) 301-8480
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Email: ecant[email protected]
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James Borneman
19
University of California
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Department of Microbiology and Plant Pathology
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3401 Watkins Drive
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Multidisciplinary Research Building Room 4130
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Riverside, CA 92521
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Phone: +1 (951) 827-3584
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Email: [email protected]
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Abstract.
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32
Herpes simplex virus 1 (HSV) is a ubiquitous human virus resident in a majority of the global
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population as a latent infection. Acyclovir (ACV), is the standard of care drug used to treat primary
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and recurrent infections, supplemented in some patients with intravenous immunoglobulin (IVIG)
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treatment to suppress deleterious inflammatory responses. We found that HSV, ACV and IVIG
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can all independently disrupt the gut bacterial community in a sex biased manner when given to
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uninfected mice. Treatment of HSV infected mice with ACV or IVIG alone or together revealed
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complex interactions between these drugs and infection that caused pronounced sex biased
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dysbiosis. ACV reduced Bacteroidetes levels in male but not female mice, while levels of the
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Anti-inflammatory Clostridia (AIC) were reduced in female but not male mice, which is significant
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as these taxa are associated with protection against the development of GVHD in hematopoietic
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stem cell transplant (HSCT) patients. Gut barrier dysfunction is associated with GVHD in HSCT
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patients and ACV also decreased Akkermansia muciniphila, which is important for maintaining
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gut barrier functionality. Cumulatively, our data suggest that long-term prophylactic ACV treatment
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of HSCT patients may contribute to GVHD and potentially impact immune reconstitution. These
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data have important implications for other clinical settings, including HSV eye disease and genital
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infections, where ACV is given long-term.
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Author Summary.
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Primary and reactivated HSV and VZV infections are treated with Acyclovir (ACV), an
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antiviral drug that blocks viral DNA synthesis. In some patients IVIG is used as adjunctive therapy
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to block deleterious inflammation. Long term preventative treatment of patients who receive stem
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transplants for various blood cancers has been successful in preventing life threatening
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reactivated HSV and VZV infections, but GVHD remains a major factor limiting transplant
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success. Studies reported here reveal that HSV infection, ACV and IVIG given alone can all
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disrupt the gut microbiota and that complex interactions between these drugs and infection results
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in even more pronounced sex biased changes in the gut bacteria community structure.
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Importantly, ACV treatment decreased the levels of specific bacterial taxa, including the anti-
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inflammatory Clostriodia and Bacteroidetes that have been shown to protect against development
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of GVHD in stem cell transplant patients. These data suggest that long term preventative
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treatment of patients with ACV may contribute to GVHD in transplant patients and have negative
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consequences in other HSV induced diseases treated long term with ACV. The health effects of
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long term ACV and IVIG treatments warrant further clinical studies.
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Introduction.
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Herpes Simplex Virus type 1 (HSV), a ubiquitous human virus is the major cause of HSV
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encephalitis (HSE), the most prevalent sporadic encephalitis resulting from either primary
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infection or reactivation of latent virus. However, despite improved diagnostic procedures and
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effective antiviral therapies, most HSE survivors have persistent neurological impairments,
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including memory and behavior disturbances, dysphasia and seizures, and only 50-65% of these
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survivors return to independent living [1, 2]. A delay in initiating Acyclovir (ACV) treatment past
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the second hospital day is associated with poor neurological outcomes [3, 4]. Recent clinical trials
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evaluating prolonged oral ACV/valaciclovir (VACV) treatment following standard 14-day
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intravenous ACV treatment reported improved neurocognitive outcomes in neonates but not
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adults for reasons that are obscure [5, 6]. Although, it is generally accepted that replication
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induced pathology underlies HSV related neurological dysfunction, supporting experimental or
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clinical evidence is lacking. Overwhelming evidence has linked inflammation to the development
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of various neurological disorders and neuropsychiatric diseases, including Alzheimer’s disease
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(AD), schizophrenia, autism spectrum disorder (ASD), multiple sclerosis (MS), Parkinson’s
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disease (PD), depression and anxiety [7-9].
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Having unequivocally established that HSE arises from exaggerated CNS inflammatory
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responses and that the immunomodulatory activities of intravenous immunoglobulins (IVIG) can
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prevent HSE in a mouse model [10], we tested the hypothesis that persistent inflammation, which
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is documented in humans and mice after HSE [11-14], causes neurobehavioral impairments in
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survivors, that should be impeded by IVIG’s anti-inflammatory activity [10]. Compared to treatment
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of HSV infected mice with ACV or PBS alone, treatment with ACV+IVIG from day 4 pi reduced
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CNS inflammation and anxiety, consistent with our hypothesis. Strikingly, development of learning
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and memory (LM) deficits that were evident only in female PBS treated mice, were inhibited by
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ACV treatment and counterintuitively, aggravated by ACV+IVIG treatment. Treatment of infected
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male mice with ACV+IVIG also impaired LM compared to ACV or PBS alone, revealing that IVIG
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antagonized the beneficial effects of ACV [15]. Intriguingly, the differential antagonistic effects of
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ACV+IVIG on cognitive behavior in HSV infected mice, compared to ACV and PBS treatment
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alone, were reflected in differential serum proteomic profiles [15]. These reported antagonistic
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effects of ACV and IVIG on LM present a conundrum, since they are at odds with the known
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mechanisms of action of these drugs.
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Rapidly accumulating evidence is revealing the critical role of the microbiome in regulating
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brain homeostasis and function such that perturbation of the gut bacteria community structure
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and function is increasingly being implicated in a variety of neurodegenerative and
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neuropsychiatric diseases. In an effort to gain insight into how HSV induces LM impairment and
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the paradoxical effects of ACV and IVIG, we investigated a role for the gut microbiota. HSV
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infection, ACV and IVIG were all associated with significant disruption of the gut bacterial
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community structure that was sex biased. Furthermore, treating HSV infected mice with either
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ACV or IVIG alone or both drugs together resulted in more pronounced sex-biased shifts in the
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gut bacterial community structure compared to uninfected mice. These results have significant
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clinical implications, particularly when patients receive prolonged ACV or IVIG treatment.
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Results.
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Equal numbers (n=8) of female and male C57BL/6 mice were bilaterally inoculated with
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virulent HSV1 strain 17+ (1x10
5
PFU/eye) by corneal scarification as previously described [15].
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At day 4 post infection (pi), ACV was administered at 1.25 mg / mouse by intraperitoneal injection
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(ip) daily for 3 days, while IVIG was given as single dose of 25 mg/mouse by ip injection on day
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4pi [15]. Fresh fecal pellets (n=1-2/ mouse) were collected on day 7 pi and stored at -80
o
C until
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processed for Illumina 16S rRNA gene sequencing to determine the effects of infection and drug
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treatment on the gut microbiome. Normal male and female mice differed in gut bacteria
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composition and unexpectedly, HSV ocular infection caused further shifts in the gut bacteria
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community and amplified this sex difference, as shown in a PCoA plot of Hellinger beta diversity
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distance values for infected compared to uninfected male and female mice (Figure 1A; P<0.05,
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Adonis Tests). In addition, HSV infection had a greater effect on gut bacterial communities in
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males (P=0.003) compared to females (P=0.011) (Figure 1A). Significant differences were
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observed at the phyla level, particularly for firmicutes (Figure 1B) with more marked differences
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evident at the species level for Clostridium aerotolerans and other clostridial species, for example
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Clostridium XIVa that ferment carbohydrates in the gut resulting in production of short chain fatty
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acids (SFCs) that contribute to barrier integrity and also exhibit anti-inflammatory properties
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(Figure 1C). A notable difference was also observed for Akkermansia muciniphila that has many
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health promoting activities, including maintaining gut barrier health (Figure 1C).
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Treating HSV infected mice with ACV from day 4 pi for three days resulted in even more
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drastic shifts in the gut bacteria composition and exaggerated sex differences (Figure 2A), than
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for infection alone. Considerable abundance changes were evident at the Phyla level for
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Bacteroidetes, Firmicutes and Verrucomicrobia (Figure 2B) and at the species level (Figure 2C).
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Notably, whereas HSV infection reduced the abundance of Firmicutes significantly in male but
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not female mice (Figure 1B), ACV reversed this effect restoring the abundance to the level in
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uninfected male mice, while also increasing the abundance in female mice (Figure 2B and Figure
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1B). Notable abundance changes at the species level included drastic suppression of Clostridium
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aerotolerans in infected male mice compared to increased abundance in females (Figure 1C),
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while ACV treatment further increased this abundance only in females (Figure 2C). Akkermansia
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muciniphila abundance was increased by infection in male mice but reduced in females (Figure
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1C), while ACV treatment resulted in total suppression of this species in female mice compared
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to a marked reduction in male mice (Figure 2C). There are many other similar changes in species
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abundance that are differentially impacted by ACV treatment in a sex-biased manner, indicative
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of complex interactions between infection, ACV effects on infected host cells, and bacteria, as
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well as metabolites produced by bacterial metabolism of ACV.
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Treatment of uninfected mice with IVIG alone also shifted the gut bacteria community
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composition with a notable marked sex effect as determined by a beta diversity analysis (Figure
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3). Males and females showed a major reduction in A. muciniphila, and a lesser reduction of
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Verrucomicrobia in males, compared to females that showed increased abundance of this phylum
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in response to IVIG treatment (Figure 4). The abundance of many other bacterial species was
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differentially altered by IVIG treatment of males and females, for example, Clostridium
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aerotolerans, Bacteroides acidifaciens and Porphyromonadaceae (Figure 4B). The response to
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IVIG was distinct in HSV infected mice, and the complex interactions between infection, ACV and
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IVIG were also evident at the phyla and species levels and were strongly sex biased as well
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(Figure 4A and 4B). IVIG treatment decreased A. muciniphila abundance markedly in infected
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males and females as did ACV, whereas in contrast, treatment with ACV+IVIG caused a notable
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increase in its abundance, indicative of antagonistic effects of these two drugs in the context of
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infection (Figure 4B) In a similar vein, C. aerotolerans abundance increased markedly in males,
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but was unchanged in females treated with IVIG, while in contrast, it was strongly decreased in
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males but slightly increased in females treated with ACV alone. In contrast, treatment with
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ACV+IVIG suppressed an IVIG-induced increase in males and an ACV-induced increase in
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females, revealing antagonism between ACV and IVIG in the context of HSV infection (Figure
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4B).
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Patients with hematologic and other malignancies have benefited immensely from
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allogeneic hematopoietic stem cell transplantation (allo-HSCT or HSCT), which can be a potent
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curative immunotherapy. However, life threatening complications such as graft-versus-host
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disease (GVHD), relapse, and infections that include reactivated HSV and VZV limit its application
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[16]. HSV and varicella zoster (VZV) reactivation has been successfully suppressed by
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prophylactic ACV treatment, though ACV-resistant (ACVr) HSV is an emerging problem [17, 18].
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Long term ACV prophylactic treatment is now routine for HSCT patients, because it was found to
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correlate with reduced HSV and ACVr HSV disease in those treated for longer than 1 year [19].
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Given this routine clinical practice, we evaluated the effects of ACV on fecal bacteria,
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because gut microbes have been implicated in GVHD pathophysiology and because we posit that
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ACV contributes to the development of GVHD by changing the gut microbiota. First, we identified
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gut bacterial changes in humans with GVHD [20-30]. Next, we determined whether the ACV-
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induced changes that we detected in this mouse study matched those GVHD-associated
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changes. Whenever we identified taxa that were altered in both types of studies, the direction of
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the change was the same, and it was consistent with our hypothesis that ACV contributes to the
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development of human GVHD by changing the gut microbiota. In the following, we describe these
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results, and we note that these ACV-induced changes were only observed in the HSV-infected
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mice and not in the uninfected mice.
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Reduced levels of several taxa belonging to the phylum Bacteroidetes have been shown
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to be associated with GVHD, indicating that these gut bacteria may play a protective role. In a
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pediatric study, GVHD patients had lower levels of the family Bacteroidaceae and the genus
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Parabacteroides [30]. In a longitudinal study, pediatric patients that had lower levels of
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Bacteroidetes prior to HSCT were more likely to develop GVHD [24]. In our study, all three if these
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taxa were reduced by ACV treatment in male but not female mice (Figure 5A).
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Reduced levels of Anti-Inflammatory Clostridia (AIC) have also been detected in human
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GVHD patients [20, 23-25, 27-30], indicating that these gut bacteria may play a protective role.
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This terminology was first introduced by Piper et al. [31] in the context of short bowel syndrome,
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and then introduced to the GVHD literature by Simms-Waldrip et al. [30]. AIC taxa include
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members of the families Clostridiaceae, Erysipelotrichaceae, Eubacteriaceae, Lachnospiraceae
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and Ruminococcaceae. In a pediatric study, decreases in Blautia and Clostridium bolteae were
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associated with the development of GVHD [30]. In an adult study, lower levels of Blautia, Blautia
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hansenii, and Blautia stercoris were associated with the development of GVHD [28]. In a
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longitudinal study, reduced levels of the Blautia before HSCT was shown to be a predictive marker
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for the development of GVHD [27]. In our study, all of these taxa were reduced by ACV treatment
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in female but not male mice (Figure 5B).
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In a more detailed analysis of AIC bacteria, we observed that while HSV infection
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increased the abundance of Blautia hansenii only in males, ACV treatment reduced its abundance
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in females but had no effect on its abundance in males (Supplemental Figure 1). Remarkably,
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a dramatic increase in B. hansenii in uninfected females was observed after IVIG treatment, and
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this increase was abrogated by ACV (compare NoHSV_F, NoHSV_IVIG_F and
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NoHSV_ACVplusIVIG_F) (Supplemental Figure 1), a result that supports sex-based differential
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effects of these drugs. However, during HSV infection, both IVIG and ACV reduced B. hansenii
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in females, whereas only IVIG reduced abundance in males. Interestingly, HSV infection
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significantly increased the abundance of the AIC genera Blautia, Allobaculum, and Clostridium
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XVIII but not Turicibacter in both males and females (Supplemental Figure 2). ACV treatment of
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HSV infected female mice resulted in significant decreases in the abundances of 4 AIC genera:
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Blautia, Allobaculum, Clostridium XVIII and Turicibacter, whereas in infected males, ACV
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decreased the abundance of Marvinbryantia and Oscillibacter (Supplemental Figure 2). In
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addition, ACV increased the abundance of Turicibacter in uninfected females but not males.
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Finally, the two most abundant operational taxonomic units (OTUs), which exhibited a
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change in their relative abundances due to ACV treatment, were assigned to the family
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Porphyromonadaceae and the species A. muciniphila (Figure 5C). While we did not find these
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taxa associated with GVHD in prior human studies, GVHD has been associated with intestinal
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barrier dysfunction [32-36]. Supporting our hypothesis that ACV contributes to the development
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of GVHD by changing the gut microbiota, members of the Porphyromonadaceae have been
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shown to cause gut barrier dysfunction [37, 38], and our Porphyromonadaceae OTU was
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increased in its abundance by ACV. In addition, A. muciniphila was decreased by ACV treatment
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in our study, and it has been shown to strengthen gut barrier functioning [39-41].
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Discussion.
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Our intention in this brief report is to alert the scientific community and especially clinicians
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to the fact that HSV infection, the antiviral drug ACV, and the immunomodulatory biological, IVIG,
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can all independently result in significant perturbations of the gut bacterial communities. Our data
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reveal complex interactions between HSV infection and ACV or/and IVIG treatment that result in
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marked alterations to gut bacterial communities. Although the clinical consequences of these
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changes have not yet been elucidated, they could have profound implications in several settings
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including HSCT-associated GVHD.
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Though the mechanisms by which ocular HSV infection causes gut dysbiosis are unclear,
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neuroinflammatory mechanisms and effects on the enteric nervous system via connected
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brainstem neuronal circuits can be envisaged [15, 42]. Indeed, recent paradigm-shifting reports
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reveal that peripheral neurons, including nociceptive and sensory neurons, can directly sense and
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respond to environmental alarms by releasing neuropeptides that can regulate immune responses
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in target organs including the gut [43, 44]. Persistence of gut dysbiosis was not evaluated here,
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but results from a behavioral study alluded to earlier suggest long-term effects of infection and
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drug treatment on gut bacterial ecology should be investigated [15]. Sex biased effects on HSV
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induced dysbiosis merit further study, as these may involve microglial responses to HSV infection
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and the microglial compartment is known to be regulated by the microbiota in a sex biased manner
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[45-47].
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The mechanism by which ACV, the standard antiviral for HSV infections, changes the gut
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microbiota likely involves its uptake into bacteria. ACV is preferentially phosphorylated by the viral
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encoded thymidine kinase (Tk) resulting in cell retention and eventual incorporation into viral DNA
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resulting in inhibition of viral replication via DNA chain termination. Because Tk is conserved in
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numerous bacterial species, ACV can be taken up and incorporated into DNA, resulting in
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bactericidal effects [48-51]. Indeed, early studies on DNA replication mechanisms relied on
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labeling bacterial DNA with tritiated thymidine and many bacterial taxa can be imaged using
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nucleoside analogues such as 1-(2_-deoxy-2_-fluoro-_-D-arabinofuranosyl)-5-[125I] iodouracil
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([125I]FIAU) that are substrates for HSV Tk [52-55]. Incorporation of [methyl-
3
H]thymidine into
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DNA has been unequivocally demonstrated for members of the Clostridium genus [56] and our
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data show ACV reduced the abundance of the Blautia genus (order Clostridiales; [57]) Blautia
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hansenii, Blautia stercoris, and Clostridium bolteae in females but not males. Additionally,
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interrogating the NCBI reference genome sequence for Blautia hansenii confirmed the presence
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of a thymidine kinase enzyme. Our data are therefore consistent with ACV causing dysbiosis by,
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at least in part, inhibiting the growth of various bacteria taxa via the Tk mechanism, though other
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mechanisms involving bacterial metabolism of ACV cannot be excluded. Clearly, the mechanisms
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by which ACV affects gut bacterial ecology are complex, which is further supported by the sex-
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biased effects.
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We also explored the effects of IVIG treatment alone and in combination with ACV in HSV-
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infected and uninfected mice, because IVIG has been used to treat HSV encephalitis (HSE) and
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is also a frontline therapy for autoimmune encephalitis, which is triggered by HSE and other insults
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[58-60]. Moreover, IVIG is being evaluated in a randomized control trial for children with all-cause
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encephalitis to determine whether neurological outcomes are improved compared to standard
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antiviral therapy alone, which is similar to our behavioral study that generated paradoxical results
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[15, 61]. Reports that IVIG’s antigenic repertoire includes reactivities to a variety of gut commensal
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antigens and metabolites have increased recently [62-64], which is consistent with a report that
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gut commensals can somehow trigger systemic IgG responses under homeostatic conditions that
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protect against systemic infection [65, 66]. We speculate that by neutralizing bacterial/host
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antigens/metabolites, IVIG is able to influence host immunity, the nervous system, and other
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physiological processes, resulting in perturbation of gut bacteria ecology. We speculate that the
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disparate and complex effects of ACV and IVIG alone and in combination on the gut bacteria
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ecology likely account for their antagonistic effects on cognitive behavior in mice latently infected
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with HSV that we alluded to earlier [15].
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This study has several limitations. Being exploratory in nature, analyses of the gut bacteria
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were done at a single time point immediately after infection or drug treatment, rather than as a
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longitudinal study that would have provided information on the persistence of the dysbiotic state
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as well as mechanistic insights as to how HSV, ACV and IVIG provoke dysbiosis. Ideally, the
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effects of ACV should be tested in latently infected mice, since virtually all HSCT patients harbor
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latent HSV. However, because HSV infection alone disrupts the gut bacterial community,
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assessing the effects of ACV on the gut bacteria community structure in the latently infected mice
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would likely be difficult. Because ACV was given ip to mice but usually orally to HSCT patients
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[67], its effects on the gut bacteria community maybe underestimated in our study.
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Notwithstanding these caveats, our finding that ACV treatment of HSV infected mice
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decreased the relative abundances of several bacterial taxa is important because these bacteria
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have been negatively correlated with the induction of and mortality from GVHD in HSCT patients
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[24, 27, 28, 30]. These results are also consistent with our hypothesis that ACV contributes to the
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development of GVHD by changing the gut microbiota. In the context of allo-HSCT, GVHD occurs
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when donor immune cells recognize recipient tissues as foreign, leading to immune-mediated
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damage to several organs and tissues including the gastrointestinal tract. This has led
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researchers to posit that the reduction of anti-inflammatory bacteria such as AIC contribute to
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GVHD pathology [30]. The results from our study extend this hypothesis to include ACV treatment
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as a putative contributor to GVHD, because ACV reduced AIC bacteria in the gut. ACV treatment
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also decreased the relative abundances of several members of the Bacteroidetes, some of which
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have been shown to exhibit anti-inflammatory properties [68-71]. More relevantly, the capsular
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polysaccharide A (PSA) from Bacteroides fragilis reduced HSV-associated mortality in mice by
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dramatically reducing immune-mediated inflammation [72]. In addition, the two most abundant
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OTUs identified in our study, whose relative abundances were positively (Porphyromonadaceae)
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and negatively (A. muciniphila) correlated with ACV treatment, have been shown to weaken [37,
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38] and strengthen [39-41] gut barrier function, respectively. These results provide an additional
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link between ACV treatment and GVHD, because barrier dysfunction, which can cause systemic
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inflammation, is a hallmark of GVHD [32-36]. Finally, long-term ACV prophylaxis initiated early
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after HSCT might also impair immune reconstitution based on results from a study of antibiotic
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depletion of gut bacteria in a murine model of syngeneic bone marrow transplantation [73]. These
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tantalizing results warrant independent validation and further detailed studies using a murine
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autologous BMT model to more rigorously evaluate the impact of long-term ACV prophylaxis on
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GVHD and engraftment, because results from such studies might eventually lead to improved
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outcomes for HSCT patients. Ideally, such future studies should be performed with mice harboring
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wild microbiota, because several recent reports show that immune responses in mice with wild
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microbiomes model human immune responses more closely than conventional mice with SPF
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microbiota [74-76].
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Materials and Methods.
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Ethics Statement
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All animal procedures were performed with prior approval of the City of Hope Institutional Animal
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Care and Use Committee (IACUC) under protocol # 07043 and within the framework of the Guide
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for the Care and Use of Laboratory Animals. C57BL6/J (B6) were bred in the vivarium at City of
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Hope.
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Mouse Studies
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Master stocks of HSV1 strain 17 composed of only of cell-released virus were prepared in
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and their titers determined on mycoplasma-free CV-1 cell monolayers. Single use aliquots of virus
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in Hanks balanced salt solution supplemented with 2% fetal bovine serum were stored at -80°C.
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Male and female mice, 6–8 weeks of age, were infected with HSV1 17
+
, a virulent strain. Mice
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were sedated with ketamine (60 mg/kg) and xylazine (5 mg/kg) prior to HSV inoculation by corneal
343
scarification. B6 mice were bilaterally inoculated with 1x 10
5
PFU per eye and monitored daily as
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previously described [15, 77].
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Administration of Acyclovir and Intravenous Immunoglobulins.
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ACV obtained from (APP Pharmaceuticals, Schaumburg, IL) was given at 50 mg/kg of
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body weight by intraperitoneal (ip) injection daily for 3 days starting on day 4 pi and PBS was
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given according to the same schedule to control mice. IVIG (Carimune, NF) obtained from CSL
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Behring (King of Prussia, PA, USA) was given ip as a single 0.5 ml dose (25 mg/mouse) on day
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4 pi or it was given in combination with a 3 day course of ACV.
353
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Illumina Bacterial 16S rRNA gene sequencing.
355
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Illumina bacterial 16S rRNA gene libraries were constructed as follows. PCRs were
357
performed in an MJ Research PTC-200 thermal cycler (Bio-Rad Inc., Hercules, CA, USA) as 25
358
µl reactions containing: 50 mM Tris (pH 8.3), 500 µg/ml bovine serum albumin (BSA), 2.5 mM
359
MgCl
2
, 250 µM of each deoxynucleotide triphosphate (dNTP), 400 nM of the forward PCR primer,
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200 nM of each reverse PCR primer, 1 µl of DNA template, and 0.25 units JumpStart Taq DNA
361
polymerase (Sigma-Aldrich, St. Louis, MO, USA). PCR primers 515F
362
(GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT) were used to
363
targeted the 16S rRNA gene containing portions of the hypervariable regions V4 and V5, with the
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reverse primers including a 12-bp barcode [78]. Thermal cycling parameters were 94°C for 5 min;
365
35 cycles of 94°C for 20 s, 50°C for 20 s, and 72°C for 30 s, and followed by 72°C for 5 min. PCR
366
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 20, 2019. ; https://doi.org/10.1101/844712doi: bioRxiv preprint
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products were purified using the MinElute 96 UF PCR Purification Kit (Qiagen, Valencia, CA,
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USA).
368
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16S rRNA gene data processing.
370
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We used the UPARSE pipeline for de-multiplexing, length trimming, quality filtering and
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operational taxonomic units (OTU) picking using default parameters or recommended guidelines
373
that were initially described in [79] and which have been updated at
374
https://www.drive5.com/usearch/manual/uparse_pipeline.html. Briefly, after demultiplexing,
375
sequences were trimmed to a uniform length of 249 bp, then filtered at the recommended 1.0
376
expected error threshold. Sequences were then dereplicated and clustered into zero-radius OTUs
377
using the UNOISE3 algorithm [80], which also detects and removes chimeric sequences; this
378
method is based on making OTUs at 100% identity. An OTU table was then generated using the
379
otutab command. OTUs having non-bacterial DNA were identified by performing a local BLAST
380
search [81] of their seed sequences against the nt database. OTUs were removed if any of their
381
highest-scoring BLAST hits contained taxonomic IDs within Rodentia, Viridiplantae, Fungi, or
382
PhiX. Taxonomic assignments to the OTUs were performed with SINTAX [82] using RDP
383
Classifier 16S training set number 16 [83] as the reference database.
384
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16S rRNA gene data analyses.
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Beta diversity was measured using QIIME 1.9.1 [84] to calculate a Hellinger beta diversity
387
distance matrix, which was depicted using principle coordinates analysis (PCoA), and statistically
388
assessed by performing Adonis tests. Statistical differences among the taxa were determined
389
using edgeR [85, 86]. Taxa relative abundance figures were made using Prism (GraphPad, La
390
Jolla, CA). Comparative analyses of the bacterial taxa between human GVHD studies and our
391
mouse study excluded sequence-selective qPCR, because the selectivity of such assays is
392
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 20, 2019. ; https://doi.org/10.1101/844712doi: bioRxiv preprint
16
questionable given the conserved nature of the 16S rRNA gene, and because the results of such
393
studies are not typically validated by sequence analyses. The bacterial sequences have been
394
deposited in the National Center for Biotechnology Information (NCBI)’s Sequence Read Archive
395
(SRA) under the BioProject Accession Number PRJNA549765.
396
397
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not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 20, 2019. ; https://doi.org/10.1101/844712doi: bioRxiv preprint
17
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not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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10.1093/bioinformatics/btp616.
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not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 20, 2019. ; https://doi.org/10.1101/844712doi: bioRxiv preprint
3.8%
3.1%
6.0%
6.5%
13.8%
48.8%
4.0%
3.8%
3.6%
3.2%
3.8%
11.3%
18.0%
38.8%
4.2%
3.7%
4.4%
5.6%
18.5%
46.6%
4.1%
8.6%
10.3%
3.7%
15.3%
43.2%
HSV_PBS_F
HSV_PBS_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
f__Porphyromonadaceae
s__Akkermansia_muciniphila
s__Bacteroides_acidifaciens
s__Clostridium_aerotolerans
f__Erysipelotrichaceae
g__Prevotella
g__Barnesiella
s__Barnesiella_viscericola
s__Alistipes_shahii
o__Clostridiales
g__Clostridium_XlVa
s__Parabacteroides_goldsteinii
g__Bacteroides
s__Barnesiella_intestinihominis
g__Alistipes
o__Burkholderiales
f__Lachnospiraceae
s__Alistipes_finegoldii
s__Bacteroides_xylanisolvens
All_least_abund
Ed_Analysis_4 Species (20 taxa)
3.8%
3.1%
6.0%
6.5%
13.8%
48.8%
4.0%
3.8%
3.6%
3.2%
3.8%
11.3%
18.0%
38.8%
4.2%
3.7%
4.4%
5.6%
18.5%
46.6%
4.1%
8.6%
10.3%
3.7%
15.3%
43.2%
HSV_PBS_F
HSV_PBS_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
f__Porphyromonadaceae
s__Akkermansia_muciniphila
s__Bacteroides_acidifaciens
s__Clostridium_aerotolerans
f__Erysipelotrichaceae
g__Prevotella
g__Barnesiella
s__Barnesiella_viscericola
s__Alistipes_shahii
o__Clostridiales
g__Clostridium_XlVa
s__Parabacteroides_goldsteinii
g__Bacteroides
s__Barnesiella_intestinihominis
g__Alistipes
o__Burkholderiales
f__Lachnospiraceae
s__Alistipes_finegoldii
s__Bacteroides_xylanisolvens
All_least_abund
Ed_Analysis_4 Species (20 taxa)
15.4%
13.8%
69.6%
8.7%
18.0%
71.8%
14.7%
18.5%
65.0%
24.8%
15.3%
57.8%
HSV_PBS_F
HSV_PBS_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
p__Bacteroidetes
p__Verrucomicrobia
p__Firmicutes
p__Proteobacteria
p__Actinobacteria
d__Bacteria
Ed_Analysis_4 Phyla (12 taxa)
20.7%
77.5%
4.7%
14.3%
79.9%
3.5%
13.6%
20.5%
62.4%
10.8%
23.5%
62.9%
16.4%
81.0%
9.7%
30.9%
58.1%
13.8%
15.4%
69.6%
18.0%
8.7%
71.8%
17.8%
16.4%
63.8%
17.0%
19.0%
62.0%
15.4%
22.9%
60.3%
16.2%
81.3%
3.8%
20.7%
7.5%
68.0%
12.9%
7.9%
78.6%
18.5%
14.7%
65.0%
15.3%
24.8%
57.8%
HSV_ACV_F
HSV_ACV_M
HSV_ACVplusIVIG_F
HSV_ACVplusIVIG_M
HSV_IVIG_F
HSV_IVIG_M
HSV_PBS_F
HSV_PBS_M
NoHSV_ACV_F
NoHSV_ACV_M
NoHSV_ACVplusIVIG_F
NoHSV_ACVplusIVIG_M
NoHSV_IVIG_F
NoHSV_IVIG_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
p__Bacteroidetes
p__Firmicutes
p__Verrucomicrobia
p__Proteobacteria
p__Actinobacteria
d__Bacteria
Ed_Analysis_6 Phyla (12 taxa)
20.7%
77.5%
4.7%
14.3%
79.9%
3.5%
13.6%
20.5%
62.4%
10.8%
23.5%
62.9%
16.4%
81.0%
9.7%
30.9%
58.1%
13.8%
15.4%
69.6%
18.0%
8.7%
71.8%
17.8%
16.4%
63.8%
17.0%
19.0%
62.0%
15.4%
22.9%
60.3%
16.2%
81.3%
3.8%
20.7%
7.5%
68.0%
12.9%
7.9%
78.6%
18.5%
14.7%
65.0%
15.3%
24.8%
57.8%
HSV_ACV_F
HSV_ACV_M
HSV_ACVplusIVIG_F
HSV_ACVplusIVIG_M
HSV_IVIG_F
HSV_IVIG_M
HSV_PBS_F
HSV_PBS_M
NoHSV_ACV_F
NoHSV_ACV_M
NoHSV_ACVplusIVIG_F
NoHSV_ACVplusIVIG_M
NoHSV_IVIG_F
NoHSV_IVIG_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
p__Bacteroidetes
p__Firmicutes
p__Verrucomicrobia
p__Proteobacteria
p__Actinobacteria
d__Bacteria
Ed_Analysis_6 Phyla (12 taxa)
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 20, 2019. ; https://doi.org/10.1101/844712doi: bioRxiv preprint
15.4%
13.8%
69.6%
8.7%
18.0%
71.8%
14.7%
18.5%
65.0%
24.8%
15.3%
57.8%
HSV_PBS_F
HSV_PBS_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
p__Bacteroidetes
p__Verrucomicrobia
p__Firmicutes
p__Proteobacteria
p__Actinobacteria
d__Bacteria
Ed_Analysis_4 Phyla (12 taxa)
20.7%
77.5%
4.7%
14.3%
79.9%
3.5%
13.6%
20.5%
62.4%
10.8%
23.5%
62.9%
16.4%
81.0%
9.7%
30.9%
58.1%
13.8%
15.4%
69.6%
18.0%
8.7%
71.8%
17.8%
16.4%
63.8%
17.0%
19.0%
62.0%
15.4%
22.9%
60.3%
16.2%
81.3%
3.8%
20.7%
7.5%
68.0%
12.9%
7.9%
78.6%
18.5%
14.7%
65.0%
15.3%
24.8%
57.8%
HSV_ACV_F
HSV_ACV_M
HSV_ACVplusIVIG_F
HSV_ACVplusIVIG_M
HSV_IVIG_F
HSV_IVIG_M
HSV_PBS_F
HSV_PBS_M
NoHSV_ACV_F
NoHSV_ACV_M
NoHSV_ACVplusIVIG_F
NoHSV_ACVplusIVIG_M
NoHSV_IVIG_F
NoHSV_IVIG_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
p__Bacteroidetes
p__Firmicutes
p__Verrucomicrobia
p__Proteobacteria
p__Actinobacteria
d__Bacteria
Ed_Analysis_6 Phyla (12 taxa)
20.7%
77.5%
4.7%
14.3%
79.9%
3.5%
13.6%
20.5%
62.4%
10.8%
23.5%
62.9%
16.4%
81.0%
9.7%
30.9%
58.1%
13.8%
15.4%
69.6%
18.0%
8.7%
71.8%
17.8%
16.4%
63.8%
17.0%
19.0%
62.0%
15.4%
22.9%
60.3%
16.2%
81.3%
3.8%
20.7%
7.5%
68.0%
12.9%
7.9%
78.6%
18.5%
14.7%
65.0%
15.3%
24.8%
57.8%
HSV_ACV_F
HSV_ACV_M
HSV_ACVplusIVIG_F
HSV_ACVplusIVIG_M
HSV_IVIG_F
HSV_IVIG_M
HSV_PBS_F
HSV_PBS_M
NoHSV_ACV_F
NoHSV_ACV_M
NoHSV_ACVplusIVIG_F
NoHSV_ACVplusIVIG_M
NoHSV_IVIG_F
NoHSV_IVIG_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
p__Bacteroidetes
p__Firmicutes
p__Verrucomicrobia
p__Proteobacteria
p__Actinobacteria
d__Bacteria
Ed_Analysis_6 Phyla (12 taxa)
5.5%
9.6%
5.6%
59.8%
3.5%
5.5%
3.7%
3.6%
6.1%
4.7%
63.7%
7.1%
3.3%
6.3%
6.6%
5.1%
13.6%
44.6%
7.9%
4.1%
5.3%
9.7%
7.9%
10.8%
38.6%
5.3%
5.9%
7.2%
59.4%
5.3%
5.6%
3.1%
17.4%
6.9%
9.7%
38.4%
3.8%
3.1%
6.0%
6.5%
13.8%
48.8%
4.0%
3.8%
3.6%
3.2%
3.8%
11.3%
18.0%
38.8%
5.1%
4.7%
4.0%
4.9%
5.1%
17.8%
44.3%
5.2%
3.5%
6.3%
6.7%
5.3%
17.0%
42.6%
8.0%
13.0%
3.8%
15.4%
43.5%
6.8%
3.2%
4.4%
16.0%
53.9%
4.1%
3.1%
4.7%
24.2%
20.6%
33.3%
4.6%
15.5%
12.9%
48.5%
4.2%
3.7%
4.4%
5.6%
18.5%
46.6%
4.1%
8.6%
10.3%
3.7%
15.3%
43.2%
HSV_ACV_F
HSV_ACV_M
HSV_ACVplusIVIG_F
HSV_ACVplusIVIG_M
HSV_IVIG_F
HSV_IVIG_M
HSV_PBS_F
HSV_PBS_M
NoHSV_ACV_F
NoHSV_ACV_M
NoHSV_ACVplusIVIG_F
NoHSV_ACVplusIVIG_M
NoHSV_IVIG_F
NoHSV_IVIG_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
f__Porphyromonadaceae
s__Akkermansia_muciniphila
s__Bacteroides_acidifaciens
s__Clostridium_aerotolerans
f__Erysipelotrichaceae
g__Prevotella
g__Barnesiella
g__Clostridium_XlVa
o__Clostridiales
s__Barnesiella_viscericola
s__Alistipes_shahii
g__Bacteroides
s__Barnesiella_intestinihominis
g__Alistipes
o__Burkholderiales
f__Lachnospiraceae
s__Parabacteroides_goldsteinii
s__Alistipes_finegoldii
s__Bacteroides_xylanisolvens
All_least_abund
Ed_Analysis_6 Species (20 taxa)
5.5%
9.6%
5.6%
59.8%
3.5%
5.5%
3.7%
3.6%
6.1%
4.7%
63.7%
7.1%
3.3%
6.3%
6.6%
5.1%
13.6%
44.6%
7.9%
4.1%
5.3%
9.7%
7.9%
10.8%
38.6%
5.3%
5.9%
7.2%
59.4%
5.3%
5.6%
3.1%
17.4%
6.9%
9.7%
38.4%
3.8%
3.1%
6.0%
6.5%
13.8%
48.8%
4.0%
3.8%
3.6%
3.2%
3.8%
11.3%
18.0%
38.8%
5.1%
4.7%
4.0%
4.9%
5.1%
17.8%
44.3%
5.2%
3.5%
6.3%
6.7%
5.3%
17.0%
42.6%
8.0%
13.0%
3.8%
15.4%
43.5%
6.8%
3.2%
4.4%
16.0%
53.9%
4.1%
3.1%
4.7%
24.2%
20.6%
33.3%
4.6%
15.5%
12.9%
48.5%
4.2%
3.7%
4.4%
5.6%
18.5%
46.6%
4.1%
8.6%
10.3%
3.7%
15.3%
43.2%
HSV_ACV_F
HSV_ACV_M
HSV_ACVplusIVIG_F
HSV_ACVplusIVIG_M
HSV_IVIG_F
HSV_IVIG_M
HSV_PBS_F
HSV_PBS_M
NoHSV_ACV_F
NoHSV_ACV_M
NoHSV_ACVplusIVIG_F
NoHSV_ACVplusIVIG_M
NoHSV_IVIG_F
NoHSV_IVIG_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
f__Porphyromonadaceae
s__Akkermansia_muciniphila
s__Bacteroides_acidifaciens
s__Clostridium_aerotolerans
f__Erysipelotrichaceae
g__Prevotella
g__Barnesiella
g__Clostridium_XlVa
o__Clostridiales
s__Barnesiella_viscericola
s__Alistipes_shahii
g__Bacteroides
s__Barnesiella_intestinihominis
g__Alistipes
o__Burkholderiales
f__Lachnospiraceae
s__Parabacteroides_goldsteinii
s__Alistipes_finegoldii
s__Bacteroides_xylanisolvens
All_least_abund
Ed_Analysis_6 Species (20 taxa)
3.8%
3.1%
6.0%
6.5%
13.8%
48.8%
4.0%
3.8%
3.6%
3.2%
3.8%
11.3%
18.0%
38.8%
4.2%
3.7%
4.4%
5.6%
18.5%
46.6%
4.1%
8.6%
10.3%
3.7%
15.3%
43.2%
HSV_PBS_F
HSV_PBS_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
f__Porphyromonadaceae
s__Akkermansia_muciniphila
s__Bacteroides_acidifaciens
s__Clostridium_aerotolerans
f__Erysipelotrichaceae
g__Prevotella
g__Barnesiella
s__Barnesiella_viscericola
s__Alistipes_shahii
o__Clostridiales
g__Clostridium_XlVa
s__Parabacteroides_goldsteinii
g__Bacteroides
s__Barnesiella_intestinihominis
g__Alistipes
o__Burkholderiales
f__Lachnospiraceae
s__Alistipes_finegoldii
s__Bacteroides_xylanisolvens
All_least_abund
Ed_Analysis_4 Species (20 taxa)
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 20, 2019. ; https://doi.org/10.1101/844712doi: bioRxiv preprint
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 20, 2019. ; https://doi.org/10.1101/844712doi: bioRxiv preprint
20.7%
77.5%
4.7%
14.3%
79.9%
3.5%
13.6%
20.5%
62.4%
10.8%
23.5%
62.9%
16.4%
81.0%
9.7%
30.9%
58.1%
13.8%
15.4%
69.6%
18.0%
8.7%
71.8%
17.8%
16.4%
63.8%
17.0%
19.0%
62.0%
15.4%
22.9%
60.3%
16.2%
81.3%
3.8%
20.7%
7.5%
68.0%
12.9%
7.9%
78.6%
18.5%
14.7%
65.0%
15.3%
24.8%
57.8%
HSV_ACV_F
HSV_ACV_M
HSV_ACVplusIVIG_F
HSV_ACVplusIVIG_M
HSV_IVIG_F
HSV_IVIG_M
HSV_PBS_F
HSV_PBS_M
NoHSV_ACV_F
NoHSV_ACV_M
NoHSV_ACVplusIVIG_F
NoHSV_ACVplusIVIG_M
NoHSV_IVIG_F
NoHSV_IVIG_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
p__Bacteroidetes
p__Firmicutes
p__Verrucomicrobia
p__Proteobacteria
p__Actinobacteria
d__Bacteria
Ed_Analysis_6 Phyla (12 taxa)
5.5%
9.6%
5.6%
59.8%
3.5%
5.5%
3.7%
3.6%
6.1%
4.7%
63.7%
7.1%
3.3%
6.3%
6.6%
5.1%
13.6%
44.6%
7.9%
4.1%
5.3%
9.7%
7.9%
10.8%
38.6%
5.3%
5.9%
7.2%
59.4%
5.3%
5.6%
3.1%
17.4%
6.9%
9.7%
38.4%
3.8%
3.1%
6.0%
6.5%
13.8%
48.8%
4.0%
3.8%
3.6%
3.2%
3.8%
11.3%
18.0%
38.8%
5.1%
4.7%
4.0%
4.9%
5.1%
17.8%
44.3%
5.2%
3.5%
6.3%
6.7%
5.3%
17.0%
42.6%
8.0%
13.0%
3.8%
15.4%
43.5%
6.8%
3.2%
4.4%
16.0%
53.9%
4.1%
3.1%
4.7%
24.2%
20.6%
33.3%
4.6%
15.5%
12.9%
48.5%
4.2%
3.7%
4.4%
5.6%
18.5%
46.6%
4.1%
8.6%
10.3%
3.7%
15.3%
43.2%
HSV_ACV_F
HSV_ACV_M
HSV_ACVplusIVIG_F
HSV_ACVplusIVIG_M
HSV_IVIG_F
HSV_IVIG_M
HSV_PBS_F
HSV_PBS_M
NoHSV_ACV_F
NoHSV_ACV_M
NoHSV_ACVplusIVIG_F
NoHSV_ACVplusIVIG_M
NoHSV_IVIG_F
NoHSV_IVIG_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
f__Porphyromonadaceae
s__Akkermansia_muciniphila
s__Bacteroides_acidifaciens
s__Clostridium_aerotolerans
f__Erysipelotrichaceae
g__Prevotella
g__Barnesiella
g__Clostridium_XlVa
o__Clostridiales
s__Barnesiella_viscericola
s__Alistipes_shahii
g__Bacteroides
s__Barnesiella_intestinihominis
g__Alistipes
o__Burkholderiales
f__Lachnospiraceae
s__Parabacteroides_goldsteinii
s__Alistipes_finegoldii
s__Bacteroides_xylanisolvens
All_least_abund
Ed_Analysis_6 Species (20 taxa)
15.4%
13.8%
69.6%
8.7%
18.0%
71.8%
14.7%
18.5%
65.0%
24.8%
15.3%
57.8%
HSV_PBS_F
HSV_PBS_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
p__Bacteroidetes
p__Verrucomicrobia
p__Firmicutes
p__Proteobacteria
p__Actinobacteria
d__Bacteria
Ed_Analysis_4 Phyla (12 taxa)
3.8%
3.1%
6.0%
6.5%
13.8%
48.8%
4.0%
3.8%
3.6%
3.2%
3.8%
11.3%
18.0%
38.8%
4.2%
3.7%
4.4%
5.6%
18.5%
46.6%
4.1%
8.6%
10.3%
3.7%
15.3%
43.2%
HSV_PBS_F
HSV_PBS_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
f__Porphyromonadaceae
s__Akkermansia_muciniphila
s__Bacteroides_acidifaciens
s__Clostridium_aerotolerans
f__Erysipelotrichaceae
g__Prevotella
g__Barnesiella
s__Barnesiella_viscericola
s__Alistipes_shahii
o__Clostridiales
g__Clostridium_XlVa
s__Parabacteroides_goldsteinii
g__Bacteroides
s__Barnesiella_intestinihominis
g__Alistipes
o__Burkholderiales
f__Lachnospiraceae
s__Alistipes_finegoldii
s__Bacteroides_xylanisolvens
All_least_abund
Ed_Analysis_4 Species (20 taxa)
3.8%
3.1%
6.0%
6.5%
13.8%
48.8%
4.0%
3.8%
3.6%
3.2%
3.8%
11.3%
18.0%
38.8%
4.2%
3.7%
4.4%
5.6%
18.5%
46.6%
4.1%
8.6%
10.3%
3.7%
15.3%
43.2%
HSV_PBS_F
HSV_PBS_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
f__Porphyromonadaceae
s__Akkermansia_muciniphila
s__Bacteroides_acidifaciens
s__Clostridium_aerotolerans
f__Erysipelotrichaceae
g__Prevotella
g__Barnesiella
s__Barnesiella_viscericola
s__Alistipes_shahii
o__Clostridiales
g__Clostridium_XlVa
s__Parabacteroides_goldsteinii
g__Bacteroides
s__Barnesiella_intestinihominis
g__Alistipes
o__Burkholderiales
f__Lachnospiraceae
s__Alistipes_finegoldii
s__Bacteroides_xylanisolvens
All_least_abund
Ed_Analysis_4 Species (20 taxa)
3.8%
3.1%
6.0%
6.5%
13.8%
48.8%
4.0%
3.8%
3.6%
3.2%
3.8%
11.3%
18.0%
38.8%
4.2%
3.7%
4.4%
5.6%
18.5%
46.6%
4.1%
8.6%
10.3%
3.7%
15.3%
43.2%
HSV_PBS_F
HSV_PBS_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
f__Porphyromonadaceae
s__Akkermansia_muciniphila
s__Bacteroides_acidifaciens
s__Clostridium_aerotolerans
f__Erysipelotrichaceae
g__Prevotella
g__Barnesiella
s__Barnesiella_viscericola
s__Alistipes_shahii
o__Clostridiales
g__Clostridium_XlVa
s__Parabacteroides_goldsteinii
g__Bacteroides
s__Barnesiella_intestinihominis
g__Alistipes
o__Burkholderiales
f__Lachnospiraceae
s__Alistipes_finegoldii
s__Bacteroides_xylanisolvens
All_least_abund
Ed_Analysis_4 Species (20 taxa)
15.4%
13.8%
69.6%
8.7%
18.0%
71.8%
14.7%
18.5%
65.0%
24.8%
15.3%
57.8%
HSV_PBS_F
HSV_PBS_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
p__Bacteroidetes
p__Verrucomicrobia
p__Firmicutes
p__Proteobacteria
p__Actinobacteria
d__Bacteria
Ed_Analysis_4 Phyla (12 taxa)
15.4%
13.8%
69.6%
8.7%
18.0%
71.8%
14.7%
18.5%
65.0%
24.8%
15.3%
57.8%
HSV_PBS_F
HSV_PBS_M
NoHSV_PBS_F
NoHSV_PBS_M
Taxa
p__Bacteroidetes
p__Verrucomicrobia
p__Firmicutes
p__Proteobacteria
p__Actinobacteria
d__Bacteria
Ed_Analysis_4 Phyla (12 taxa)
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 20, 2019. ; https://doi.org/10.1101/844712doi: bioRxiv preprint
HSV_F
HSV_ACV_F
HSV_M
HSV_ACV_M
NoHSV_F
NoHSV_ACV_F
NoHSV_M
NoHSV_ACV_M
0
5
10
15
20
Relative Abundance %
Bacteroidaceae
HSV_F
HSV_ACV_F
HSV_M
HSV_ACV_M
NoHSV_F
NoHSV_ACV_F
NoHSV_M
NoHSV_ACV_M
0
2
4
6
Parabacteroides
Relative Abundance %
HSV_F
HSV_ACV_F
HSV_M
HSV_ACV_M
NoHSV_F
NoHSV_ACV_F
NoHSV_M
NoHSV_ACV_M
0
1
2
3
4
5
P. g o l d s t e i n ii
Relative Abundance %
HSV_F
HSV_ACV_F
HSV_M
HSV_ACV_M
NoHSV_F
NoHSV_ACV_F
NoHSV_M
NoHSV_ACV_M
0.0
0.1
0.2
0.3
0.4
Relative Abundance %
Blautia
HSV_F
HSV_ACV_F
HSV_M
HSV_ACV_M
NoHSV_F
NoHSV_ACV_F
NoHSV_M
NoHSV_ACV_M
0.00
0.05
0.10
Relative Abundance %
Blautia hansenii
HSV_F
HSV_ACV_F
HSV_M
HSV_ACV_M
NoHSV_F
NoHSV_ACV_F
NoHSV_M
NoHSV_ACV_M
0.00
0.02
0.04
0.06
Relative Abundance %
Blautia stercoris
HSV_F
HSV_ACV_F
HSV_M
HSV_ACV_M
NoHSV_F
NoHSV_ACV_F
NoHSV_M
NoHSV_ACV_M
0
10
20
Relative Abundance %
A. muciniphila
HSV_F
HSV_ACV_F
HSV_M
HSV_ACV_M
NoHSV_F
NoHSV_ACV_F
NoHSV_M
NoHSV_ACV_M
0
10
20
30
40
Relative Abundance %
Porphyromonadaceae
HSV_F
HSV_ACV_F
HSV_M
HSV_ACV_M
NoHSV_F
NoHSV_ACV_F
NoHSV_M
NoHSV_ACV_M
0.00
0.02
0.04
0.06
0.08
Relative Abundance %
Clostridium bolteae
not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which wasthis version posted November 20, 2019. ; https://doi.org/10.1101/844712doi: bioRxiv preprint