Humoral Immune Responses to HIV in the Mucosal Secretions and Sera of HIV-Infected Women

Abstract

Although sera and all external secretions contain antibodies to HIV, their levels, specificity, isotypes, and relevant effector functions display a great degree of variability. Antibodies that bind HIV antigens and neutralize the virus are predominantly associated with the IgG isotype in sera and in all external secretions, even where total levels of IgG are much lower than those of IgA. Rectal fluid that contains high IgA, but low IgG levels, displayed low neutralizing activity independent of antibodies. Therefore, external secretions should be evaluated before and after selective depletion of Ig. At the systemic level, HIV-specific IgA may interfere with the effector functions of IgG, as suggested by recent studies of individuals systemically immunized with an experimental HIV vaccine. Although HIV-specific IgG and IgA antibodies may exhibit their protective activities at mucosal surfaces through interference with viral entry and local neutralization at the systemic level, such antibodies may display discordant effector functions.

Introduction

Although the majority of bacterial and viral infections, including HIV, enter the body through the large surface areas of mucosal membranes, the ensuing humoral and cellular responses are most frequently determined in sera and in lymphocytes isolated from peripheral blood; immune responses in mucosal secretions and tissues are usually not evaluated. In view of the considerable independence of the systemic and mucosal compartments of the immune system, with respect to the immunoglobulin (Ig) isotypes and tissue origin of lymphocytes, the evaluation of systemic responses may not reflect the quality and magnitude of immune responses generated at the site of entry of infectious agents. Furthermore, individual mucosal sites display marked differences with respect to the dominant Ig isotypes and effector functions involved in their protective activities, as well as the phenotypes and origin of B and T cells resident in mucosal tissues.^1–3 These facts are particularly relevant to HIV infections. The mucosae of the genital and gastrointestinal tracts are the most commons sites of viral entry through heterosexual and homosexual encounters. Importantly, these two compartments display highly significant differences in the total levels of IgA and IgG and their molecular forms, numbers and isotypes of antibody-secreting cells (ASC), expression of Ig-transporting receptors on epithelial cells, and local vs. systemic origin of antibodies. The presence of mucosal inductive sites, expression of homing receptors on lymphocytes and corresponding ligands on endothelial capillary cells, and strong hormonal influence on the total levels of Ig in the female genital tract during the menstrual cycle are also characteristic of these two compartments.^1–4

The purpose of this review is to critically discuss problems encountered in the evaluation of immune responses in external secretions, emphasize the unexpected dominance of HIV-binding as well as neutralizing antibodies of the IgG isotype in sera and all external secretions examined, and to identify current controversial issues encountered in the evaluation of HIV-specific responses in mucosal secretions.

Problems encountered in the evaluation of humoral immune responses in external secretions of HIV-infected individuals

There are no uniformly accepted mucosal collection and specimen processing methods that would allow for the generation of comparable results from individual laboratories, despite attempts to standardize these procedures.⁵ Although the pronounced dominance of secretory IgA (S-IgA) was observed in almost all secretions irrespective of the collection procedure, the total levels of S-IgA, and especially of IgG, in female genital tract secretions display enormous differences during the menstrual cycle.^4–7 All external secretions contain Ig at much lower levels than those in serum and display enormous variabilities in their concentrations, which is true even for the same type of secretion (e.g., genital tract fluids).⁵ This is partially due to different collection and sample processing methods, dilution with lavage fluids, increased flow rates upon inadvertent stimulation, sensitivity to bacterial and endogenous proteases, and binding to other proteins and glycoproteins, such as mucin, and the tendency of Ig to form aggregates which interfere with precise quantitation.⁵

ELISA provides reliable information with respect to the HIV-specific antibodies of the IgG, but not IgA isotypes. This point was convincingly demonstrated in two extensive comparative evaluation studies of rectal or cervico-vaginal lavage fluids (RL and CVL, respectively) performed in six different laboratories.^8,9 Using well-established assays, there was a remarkable concordance of results with respect to the IgG HIV-specific antibodies. In contrast, marked differences were obvious in the positivity detection, as well as the levels of antibodies of the IgA isotype. This may be partly due to the differences in antigens used for plate coating and/or antibodies used for the development of ELISA. Furthermore, frequent false-positive results were reported earlier for the measurement of HIV-specific IgA antibodies in external secretions.¹⁰

Enhanced-chemiluminescence western blot assay (ECL-WB) has been used in several studies.^8,9,11–15 Due to the high sensitivity and the ability of antibodies to react with various HIV-derived antigens, ECL-WB has generated highly reliable results for the detection of HIV-specific antibodies of both IgG and IgA isotypes. Although not quantitative, the frequency of detection was higher than that observed by ELISA.

Virus neutralization (VN) assessment in external secretions presents several unavoidable problems, including: low content of total Ig,⁵ limited volumes of obtained fluids which have to be used at low dilutions,^9,11,14 the late appearance of VN antibodies after HIV infection,^16–21 as well as the presence of innate humoral factors (e.g., secretory leukocyte protease inhibitor, lactoferrin and others^22–25). Therefore, a careful choice of viruses to be used and the selective removal of IgG and IgA is required to ascertain that the VN is indeed mediated by antibodies.¹⁴ Furthermore, serum IgG and IgA HIV-specific antibodies exhibited different patterns and variability with respect to the stage of HIV infection, including acutely infected patients, elite controllers, long term non-progressors, AIDS, and patients on HAART.²⁰

In addition to differences in specificity of antibodies for Env proteins and peptides,^8,9,21 glycans associated with HIV antigens need to be also considered. HIV gp120 is a heavily glycosylated outer component of envelope glycoprotein (Env) trimers, with ~50% of its molecular mass contributed by N-linked glycans, which are involved in the binding to the host-cell receptor and co-receptor(s) and the initial steps of cell entry and infection.²⁶ Another aspect of Env glycosylation is related to the antibody binding; gp120 glycans serve as epitopes for some antibodies and as shields against other VN antibodies.²⁷ HIV-1 escape variants that emerge due to the pressure of the immune system during the chronic infection exhibit diverse env sequences.²⁸ Recent studies identified transmitted “founder” virus (TFV) genome sequences and revealed that in most cases the infection starts from transmission of a single virus or few viruses.^{29, 30} Env sequences from TFV and chronic-stage virus (CSV) often differ in the number and localization of potential N-glycosylation sites (PNGS); it has been speculated that these new PNGS may generate a shield against VN antibodies.³¹ The differential cell-specific glycosylation of gp120 affects recognition by HIV-1-specific antibodies.^32,33 It is well-established that variable PNGS on Env gp120 are a characteristic of escape variants of HIV-1. Notably, some of the VN antibodies, such as PG9 and PG16, recognize Env glycopeptides in the context of specific glycosylation of some sites.^32,33 Thus, it is important for the assays concerning measurement of virus-binding, as well as VN antibodies, to use well-defined and well-characterized Env or viruses with these Env variants. In view of the fact that antibodies in sera and external secretions may differ in their strict antigen specificity patterns,³⁴ it is obvious that experiments need to be performed using sera as well as external secretions.

HIV-specific humoral responses in sera and all external secretions: Dominance of IgG

A. HIV-binding antibodies

Mucosally acquired infections or mucosal immunizations induce antigen-specific humoral immune responses dominantly of the IgA isotype at the site of infection or immunization, and in secretions of anatomically remote mucosal tissues, due to the dissemination of precursors of ASC of the IgA isotype through the common mucosal immune system.^35–37 Based on the dominance of S-IgA in almost all external secretions, many studies focused on the detection of HIV-specific antibodies of the IgA isotype.^1,18,38,39 However, subsequent quantitative evaluations clearly demonstrated that in contrast to the pronounced S-IgA responses induced by the majority of bacterial and viral mucosal infections,^40,41 HIV responses are represented by specific antibodies of the IgG isotype.^{1,8,9,11,12,14,,42–44} Surprisingly, the dominance of IgG HIV-specific antibodies was obvious even in secretions in which S-IgA constitutes 95% or more of total Ig (such as the intestinal fluid and saliva).^12,15 This marked dominance of HIV-specific IgG was detected irrespective of the differences in ELISA protocols and HIV antigens used.^8,9,15 The evaluation of the IgG subclass-association of HIV-specific antibodies demonstrated their restriction to the IgG1 and IgG3.⁴⁴ Interestingly, the calculations of “specific antibody activities” (HIV-specific vs. total IgA or IgG antibodies) in various external secretions and also in sera clearly indicated that these values are not identical for all secretions and display marked, site-specific differences.^12,15,44 These findings suggest that the HIV-specific antibodies may originate both from the local synthesis in individual mucosal tissues, and from a highly variable plasma contribution.^12,15,44 Based on such approaches, a significant local production of HIV-specific IgG1 antibodies was convincingly demonstrated for CVL.⁴⁴ Using ELISA, HIV-specific antibodies of the IgA isotype were present at low levels in the majority of samples or even absent in others.^8,9,11,12,15 However, the levels of total IgA in these secretions were comparable or even higher than in those collected from non-infected individuals.^8,9,12,14,15 Although present in almost all plasma/serum samples, the levels of HIV-specific IgA antibodies displayed extremely high variability and were low in comparison to IgG (for example, median values for 50 individuals were 3,290 ng/ml for IgA vs. 108,000 ng/ml for IgG, respectively).¹⁵ The low levels of HIV-specific IgA in all external secretions were also observed in serum samples, suggesting that HIV in humans and SIV in macaques do not elicit pronounced IgA responses in mucosal or in systemic compartments of the immune system.^8,9,12,15,45 When present, HIV-specific IgA is restricted in sera and saliva to the IgA1 subclass.³⁹

Enumeration of ASC in peripheral blood of HIV-infected individuals corroborated serological data.¹² Irrespective of the route of acquisition, length of infection, CD4⁺ cell counts, and viral loads, ASC specific for HIV Env gp120 or gp160 of the IgG isotype were present in statistically significant higher numbers (0.71% or 1.24%, respectively) than those of the IgA (0.15% or 0.34%) or IgM (0.45% or 0.55%) isotypes.¹² In the intestinal mucosa of jejunum, ileum and rectum, IgA ASC greatly outnumbered those producing IgG. However, when ASC of the IgG vs. IgA specific for gp160 and gp120 were enumerated as the percentage of total IgG or IgA ASC, it was obvious that in the intestine, 0.2–1.2% of IgG ASC were specific for Env antigens, while only 0.02–0.25% ASC were of the IgA isotype (Moldoveanu, unpublished results). In other studies,^46,47 a marked increase in total ASC of all isotypes was observed in the intestinal mucosal of HIV-infected individuals, apparently due to the polyclonal B cell activation.⁴⁷ A higher relative frequency of ASC specific for gp160 in the IgG than the IgA isotype were observed.⁴⁶

B. HIV-neutralizing antibodies

The protective effect of HIV-neutralizing antibodies has been demonstrated in vitro and in vivo in extensive studies performed in macaques.^48–51 Therefore, the induction of such antibodies is one of the most important current efforts in HIV vaccinology.^19,52,53 In the majority of studies, VN antibodies have been evaluated in the sera of HIV-infected or immunized individuals. As discussed above, the determination of VN in mucosal secretions is complicated by a number of unavoidable problems. VN antibodies are usually induced at later stages of infection and in comparison to binding antibodies, reach low levels.^1,17,19–21 With the exception of CVL and semen, in which total IgG represents the dominant Ig isotype, other secretions contain IgA at concentrations that are much higher than those of IgG.⁵ In view of the fact that the HIV-binding antibodies are mainly associated with the IgG isotype, the evaluation of VN in some secretions (e.g., RL fluid) is difficult.¹⁴ Furthermore, the anti-viral activity of external secretions may be also mediated by many factors of innate humoral immunity, which may mask or interfere with antibody-dependent VN (see above). To avoid this problem, VN should be performed before and after selective removal of IgG and/or IgA from the secretion examined.¹⁴ This approach is feasible in contrast to the evaluation of Ig isolated from RL or CVL. The low levels of total and especially HIV-specific VN antibodies and the partial loss and denaturation of Ig molecules during desorption from affinity gels (low pH, high salt concentration, etc.,), contribute to the difficulties encountered with a reliable determination of VN activity of Ig isolated from CVL and RL. In HIV-infected individuals’ sera and external secretions, represented by CVL, VN antibodies were associated dominantly with the IgG isotype.^9,14 This conclusion was confirmed by the selective removal of IgG: the VN became greatly reduced or even undetectable.¹⁴ In contrast, selective removal of IgA from sera reduced but did not abolish VN activity, indicating that IgA VN antibodies are present, but at levels much lower than those of IgG.¹⁴ In contrast to CVL, evaluation of VN in RL collected in parallel yielded results indicating that VN was mostly mediated by innate humoral factors rather than Ig: selective removal of IgG and/or IgA did not substantially alter the low level of VN observed in RL samples.¹⁴ It should be emphasized that in this as well as many previous studies, properly collected RL contain (in contrast to CVL) only trace amounts of IgG⁵, the dominant isotype associated with VN, and the IgA present in RL in relatively larger quantities does not exhibit VN activity.¹⁴ However, VN IgA antibodies have been detected in external secretions by some, but not all investigators (for review see¹). Nevertheless, it is generally agreed that HIV-specific antibodies of the IgA isotype, which in vitro display effector functions desirable for the protection against HIV infection, including VN,^1,38,54–58 are not regularly induced by HIV infection or immunization with experimental HIV vaccines.¹

Current controversies and future directions

Although the evaluation of humoral responses in external secretions of genital and intestinal tracts of HIV-infected or vaccinated individuals is compromised by the above-described difficulties, the importance of a parallel evaluation of humoral responses in plasma/sera and relevant secretions is justified by the infrequently appreciated mutual independence in magnitude and quality of immune responses induced in the systemic and mucosal compartments.^35–37 The dominant Ig isotype in most external secretions, S-IgA, is derived almost exclusively from the local production and selective, receptor-mediated transport into external secretions.⁵⁹ Several comparative studies clearly indicated the independence of IgA present in plasma and external secretions with respect to the maturation patterns, molecular forms and particularly to effector functions, some of which are highly relevant to HIV infection. The protective function of HIV-specific IgA in mucosal protection has been demonstrated in vitro by its extracellular and intracellular VN activity, inhibition of HIV uptake by epithelial cells, and exclusion of HIV-IgA immune complexes from the epithelial cells.^{1,3,38,54–58,60} Unexpectedly, and in sharp contrast to this demonstrable protective effect of S-IgA, the induction of HIV Env-specific IgA responses in plasma of volunteer systemically immunized with an experimental HIV vaccine correlated directly with the higher rate of HIV infection.⁶¹ Although the reasons for this this unexpected finding have not been elucidated, there are several potential mechanisms involved. It is possible that IgA bound to HIV interferes with effector functions of IgG including VN, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated viral inhibition (ADCVI), and protection of HIV-infected cells from immunologically-mediated elimination (see Tables I). This finding underlines underappreciated differences in effector functions of IgA in mucosal and systemic compartments.⁶² In HIV infection, it appears that strong IgA responses in the systemic compartment may have detrimental effects, in contrast to the protective functions of S-IgA in secretions of the genital and intestinal tract (Table I). The potential mechanisms involved in the suppression of IgA and IgG2 HIV-specific antibodies were recently elucidated and are apparently mediated by the interaction of HIV-infected dendritic cells, producing Nef, which upon contact with relevant B cells, selectively inhibit their differentiation and production of IgA and IgG2 antibodies.^{63, 64}

Table I.

Comparative effector functions of HIV-specific antibodies of IgG and IgA isotypes in the systemic and mucosal compartment of the immune system

Compartment	IgG	IgA
SYSTEMIC	Present in all infected individuals	Present in the majority of infected individuals at low levels
Plasma/serum	VN ADCC ADCVI	VN? Possible blocking of functions (VN, ADCC, ADCVI) mediated by IgG “Protection” of the virus and/or virus-infected cells
MUCOSAL
Genital tract	Dominant Ig isotype	Low or absent IgA anti-HIV responses
	HIV-binding and VN	VN in some secretions
	Protective effect of antibodies	Inhibition of virus attachment
	ADCC in mucosal tissues?	Intracellular neutralization
	pH-dependent, FcRn-mediated IgG-HIV uptake	HIV-IgA excretion
Intestinal tract	Low levels	Low or absent HIV-specific IgA antibodies
	VN difficult to detect	Excretion of HIV-IgA

The levels and spectra of antigen-specific antibodies, and effector functions of IgG antibodies in plasma/serum and external secretions also display marked differences (Table II), some of which may be relevant to HIV-specific humoral responses. In the macaque models, systemically or vaginally administered monoclonal VN HIV-specific antibodies of the IgG isotype protected animals against intravaginal SHIV challenge.^48–51 Effective transport of IgG from the circulation and also from local production in the genital tract^65,66 is mediated by the FcRn receptor expressed on epithelial cells.⁶⁵ Most recent in vitro generated results indicate that the FcRn-mediated and pH-dependent transport of IgG also enhances transcytosis of IgG-bound HIV across intact monolayers of epithelial cells of genital, as well as intestinal origin.⁶⁷ Interestingly, the FcRn-mediated transport as well as IgG-dependent ADCVI are influenced by the glycosylation pattern of IgG in that only fully glycosylated molecules react with corresponding cellular receptors; alterations and deficiencies of terminal glycan residues result in the diminished receptor reactivity.^68,69 Consequently, the vaginal pH and the glycosylation pattern of IgG may play an important role in the protection or enhanced acquisition of HIV infection. Based on the marked immunological differences in the systemic and mucosal compartments, as well as the unique immunological characteristics of the genital and intestinal tract, with respect to the magnitude, quality, and duration and Ig isotype-dependent effector functions, the mucosal humoral immune responses should be evaluated in parallel with responses in the systemic compartment.

Table II.

Paucity of HIV-specific IgA responses in HIV-infected individuals

Fluid	Isotype	Total level (in µg/ml) means	Frequency of HIV+ (in %)		HIV Env- specific (in µg/ml)	Specific/ Total (in %)	Virus neutralization (in %)
			ELISA	WB
		15,513	100	100
	IgG	18,632	100	100	108	0.7
Serum		28,128	100	100			98–100
		2,173	98	94
	IgA	2,405		94	3.2	0.15
		2,024	8–100
	IgG	107.9	100	100
Cervicovaginal lavage (CVL)		47	100		3.1	2.9
		62.2	100	100			7.7–38.5
		31.3	81	100
	IgA	6		100	0.07	0.2
		7.4	0–30	42
		4.5	86	82
Rectal lavage (RL)	IgG	8	82		0.1	2.2
		9.4	62	81
		164.9	14	73
	IgA	72		73	0.03	0.02
		129	6	7.7