Natural autoantibodies in healthy neonatals recognizing a peptide derived from the second conserved region of HIV-1 gp 120 Prirodna antitela prisutna kod zdrave novoro đ en č adi koja prepoznaju peptid poreklom iz drugog konzerviranog regiona HIV-1 gp 120

Background/Aim. High sera reactivity with a peptide derived from human immunodeficiency virus HIV-1 envelope protein gp120, NTM1, correlate with non-progressive HIV-1 infection and also may have protective role in breast and prostate cancer. We also detected a low NTM1 reactive antibodies titer in healthy HIV negative sera and showed that antibody levels can be significantly increased with vigorous physical activity. However, the immune system seems to be unresponsive or tolerant to this peptide, implicating that the NTM1 sequence encompasses or overlaps a certain innate immune epitope. The aim of this study was to present evidences that NTM1 – binding antibodies – are components of innate immune humoral response, by confirming their presence in sera of newborn babies. For this purpose we collected a set of 225 innate antigen sequences reported in the literature and screened it for candidate antigens with the highest sequence and spectral similarity to NTM1 derived from HIV-1 gp120. Methods. Sera from 18 newborns were tested using ELISA, with peptide NTM1. Sequences from innate antigen database were aligned by an EMBOSS Water bioinformatics tool. Results. We identified NTM1 reactive antibodies in sera of HIV negative newborn babies. Further, in order to identify which of already known innate antigens are the most similar to NTM1 peptide we screened innate immune antigen sequence database collected from the literature. This screening revealed that the most similar sequence are ribonucleoproteins RO60, in addition to previously identified Nterminus of vasoactive intestinal peptide. Conclusion. The results of this study confirm the hypothesis that NTM1 recognizing antibodies are a part of humoral innate immune response. Further, computational similarity screening revealed a vasoactive intestinal peptide and RO60 as the most similar sequences and the strongest candidate antigens. In the light of the presented results, it is appealing that testing blood reactivity at birth, with specific innate antigens, particularly a vasoactive intestinal peptide, can reveal the potential to develop or boost protective immune response in breast and prostate cancer and HIV infection later in life.


Introduction
In spite tremendous progress that has been made by introducing highly active antiretroviral therapy (HAART) in inhibiting HIV-1 virus through disrupting reverse transcription, integration or proteolytic processing of viral proteins, some important problems as persistent viral reservoirs, drug resistance and toxicities still remain.HAART can effectively keep the viral replication at an undetectable level, prolonging the life expectancy of the infected and reducing the viral transmission.However, several host and virus factors can slow down and block the disease progression [1][2][3][4] and also, various immunological factors 5,6 might play an important role.NTM1 peptide, derived from the C-terminus of the second conserved region of HIV-1 envelope protein gp120, and anti-NTM1 antibodies have been suggested to be important in controlling HIV disease.These antibodies have been significantly prevalent in sera of long-term nonprogressors (LTNP), the HIV positives that are capable of keeping viral load bellow 10 000 copies/mL without any retroviral therapy for more than 10 years.The same is observed in patients in asymptomatic phase of the HIV-1 disease 7 .NTM1 binding antibodies are reported in a small number of healthy individuals and extremely high titer values are detected in elite athletes 8 .Although the immune system seems to be unresponsive or tolerant to this peptide [9][10][11] , the titer NTM1 recognizing antibodies can be increased by continuous and vigorous exercising 12 .Taken together these data implicate that NTM1 peptide encompasses or overlaps the innate immune epitope sequence involved in cellular pathway activated by physical activity.Based on the sequence similarity and in vitro cross-reactivity we previously hypothesized that the candidate binding antigen for these was vasoactive intestinal peptide (VIP), the pleiotropic extracellular molecule important in many physiologic functions, including glucose homeostasis, neuroprotection, memory, gut function, modulation of the immune system and circadian function.Due to its important immunomodulatory and neuromodulatory activities, circulating levels of VIP are under tight control.Natural anti-VIP autoantibodies are potent modifiers of its biological actions and important regulators of its circulating level [13][14][15][16][17] .
Natural autoantibodies (NAbs) are an important component of the immune system, existing in all vertebrates and demonstrating a non-pathogenic anti-self reactivity 18 .NAbs are reactive with only a restricted and specific set of proteins 19 .The function of these antibodies, although not fully elucidated, is to provide early innate immune protection against certain patho-gens, as well as the removal of possible autoantigens through scavenging dead or apoptotic cellular debris through the lifetime (for review see Lutz et al. 20 ).In general, mediators of innate humoral immunity are low-affinity polyreactive antibodies with an ability to bind to diverse, similar epitopes.
The aim of this study was to present evidences that NTM1-binding antibodies are components of innate immune humoral response, by confirming their presence in sera of newborn babies.For this purpose we collected a set of 225 innate antigen sequences reported in the literature and screened it for candidate antigens with the highest sequence and spectral similarity to NTM1 derived from HIV-1 gp120.Computational similarity screening revealed VIP and a ribonucleoprotein, RO60, as the most similar sequences and the strongest candidate antigens, although other highly similar sequences were also identified.These results imply that testing blood reactivity with specific innate antigens at birth can reveal potential to develop or boost protective immune response in breast and prostate cancer and HIV infection.

Sequences
The HIV1 gp120 NTM1 sequence was as in paper of Djordjevic et al. 21Innate immune antigen sequences are listed in Addendum 1.

Sequence alignments
Sequence alignments are calculated by an EMBOSS Water, a tool that uses the Smith-Waterman algorithm to calculate the local alignment of two sequences 22 .Sequences with scores higher than 20 (scores were from 7 to 23) are selected as candidate antigens.

Human subjects
Sera were collected from 9 preterm and 9 term newborns within the first 7 days after birth.The preterm infants were born between the weeks 27-34 of gestation, with the weight 950-2,200 g.The term newborns were born after 37 weeks of gestation, with the weight more than 2,500 g.After centrifugation, sera were collected and stored at -

ELISA
ELISA was performed with peptide (NTM1)4-SOC4 by the following procedure: polystyrene microtiter plates (Greiner, Germany) were incubated overnight at 4°C with 100 l of peptides (1.25 g/well) diluted in carbonate buffer, pH 9.6.Plates were washed with phosphate-buffered saline (PBS)-0.05%Tween and non-specific sites were blocked with 200 l PBS containing 5% bovine serum albumin (BSA) for 2 h at room temperature.After 6 washings, serum specimens were added to the wells (100 l /well).Sera were diluted 1 : 20 in 5% BSA in PBS.Plates were incubated for 4h at room temperature.After 6 washings with PBS-0.05%Tween, 100 l of goat anti-human IgM alkaline phosphataseconjugated antibodies (Sigma), diluted 1 : 50 were added and the plates were incubated for 30 minutes at room temperature.After 6 washings, p-nitrophenyl phosphate (pNPP) substrate was added and the absorbance optical density (OD) measured at 405-620 nm after 15 minutes.Each sample was tested twice.As a control we used antigen from Serion ELISA classic Cytomegalovirus IgM (Institute Virion\Serion GmbH).

Statistical analysis
The significance of the differences in O.D. values for preterm and term infants was calculated by Student's t-test, as the sample sizes were relatively small.The 2 groups were unpaired with uneven variance and therefore they were considered as part of a two-tailed, heteroscedastic matrix.

Results
In order to show that NTM1 binding antibodies are innate natural self-binding antibodies we determined their presence in sera of HIV-1 negative neonates.Our previous studies showed the presence of NTM1 antibodies of IgG subclass in HIV positive LTNPs, as well as in elite athletes and in small percentage of healthy HIV-adult population 7 , 23 , but here NTM1 binding of IgM subclass of antibodies specific to neonatal population were measured.Maternal antibodies of IgG subclass actively transfer across the placenta and therefore can be related to her adaptive immune response 24 .Reactivity of sera in a small cohort of 18 HIV-1 negative new born babies (Table 1) was determined by the ELISA immunoassay (Figure 1).Statistical analysis revealed that this reactivity in sera from preterm neonates was significantly higher in comparison with the reactivity of term neonates (p < 0.001).In search for innate epitopes similar to NTM1 we screened literature data and identified 225 protein sequences reported to encompass innate immune epitopes (Addendum 1) 20, . Consiering the ability of natural antibodies to bind to diverse epitopes we selected the set candidate antigens as described in Methods section (Table 2).

Discussion
Certain types of autoreactive immune cells and antibodies are common in healthy individuals and play an important role in body homeostasis.Several functions have been assigned to NAbs: neutralization of microbes and microbial toxins as natural first-line defence against infection, removal of senescent/altered self molecules and cells and immunomodulation and immunosignaling.New and unexpected insights into the functional roles of NAbs are still emerging, especially regarding their protective functions.NAbs are encoded by V(D)J genes in germline configuration and belong to IgG, IgM and IgA subclasses.
Several studies which investigated humoral immune response associated with the control of HIV-1 disease progression showed that the antibodies recognizing the peptide derived from C-terminus of the second conserved region of HIV-1 gp120, NTM1, correlate with non-progressive HIV-1 infection.Recently, the potential protective role of NTM1 antibodies has also been shown in breast and prostate cancer patients.Due to the fact that the peptide NTM1 is not immunogenic in  humans, it has been suggested that the antibodies recognizing this peptide represent natural autoantibodies.Thus, we have confirmed this hypothesis by detecting NTM1 antibodies in newborn babies.Sera samples of a small cohort of 18 newborns not older than 7 days tested in ELISA experiments showed a significant binding to NTM1 peptide.The difference between preterm and term serum titers are in line with already observed major differences of functional immune components between these two categories of neonates as reviewed in Sharma et al. 48.This may be explained by specific innate immune defense pathways in protecting preterm infants against infection, or by deregulated innate immune responses which play a major role in the etiology of certain preterm neonatal complications later in life.Further, assuming that NTM1 recognizing antibodies belong to the majority of NAbs displaying rather low affinity and polyreactivity for a range of ligands 49 we scanned the set of innate antigen sequences in order to identify those that are most similar to NTM1.The collection of the 225 tested protein sequences from literature data represents significant, but far from exhaustive list of innate immune antigens (Addendum 1).Based on this inquiry we selected 10 most similar sequences as potential binding antigens for NTM1 recognizing antibodies (Table 2).The two prominent candidate antigens which show the highest similarity score to NTM1 are extracellular VIP, whose sera titer is tightly regulated by NAbs and RO60, which is an antigen for anti-RO60 antibodies involved in the clearance of apoptic debris 50 .These findings are in perfect agreement with major functional roles attributed to the natural IgM antibodies in the maintenance of tissue homeostasis and immune regulation [51][52][53] .However, we suggest that more probably VIP serves as the most important self-binding molecule for these antibodies for the following reasons: it was identified as one of the most important nodes in IgM antigen network conserved in both, mothers and babies 54  healthy adults 55 , and the levels of circulating VIP in newborns are significantly prevalent in preterm neonates compared to term born babies 56 which could explain the statistically significant prevalence of NTM1 recognizing antibodies (p < 0.001) in our study.

Conclusion
The information about the presence of NTM1 recognizing natural antibodies at birth may be of significant importance later in life due to the fact that these antibodies might have protective roles in HIV-1 disease and in breast or prostate cancer.Previous findings that these NAbs are retained throughout life with potential of sera titer to be sig-nificantly increased by physical exercise emphasize the need for future exploration of NTM1 recognizing antibodies as personalized therapeutic strategy based on the natural antibodies repertoire.In the light of the presented results, it is appealing that testing blood reactivity at birth, with specific innate antigens, particularly vasoactive intestinal pephole (VIP), can reveal the potential to develop or boost protective immune response against certain life threatening diseases.
20 C. The sample material used in this study was what remained of serum used for standard biochemical testing.Blood specimens were not collected specifically for these experiments.The study was approved by the Ethics Committee of the Institute for Neonatology, Belgrade, Republic of Serbia.Djordjevi Vuji i A, et al.Vojnosanit Pregl 2014; 71(4): 352-361.
Fig. 1 -ELISA results of sera reactivity in the preterm and the term neonates.Antibodies recognizing peptide NTM1 were significantly more prevalent in serum samples from the preterm neonates compared to the term neonates (p < 0.001).