Pre-existing neutralizing antibodies prevent CD8 T cell-mediated immunopathology following respiratory syncytial virus infection

Despite being a leading cause of severe respiratory disease, there remains no licensed respiratory syncytial virus (RSV) vaccine. Neutralizing antibodies reduce the severity of RSV-associated disease, but are not sufficient for preventing reinfection. In contrast, the role of memory CD8 T cells in protecting against a secondary RSV infection is less established. We recently demonstrated that high-magnitude memory CD8 T cells efficiently reduced lung viral titers following RSV infection, but induced fatal immunopathology that was mediated by IFN-γ. To evaluate the ability of RSV-specific neutralizing antibodies to prevent memory CD8 T cell-mediated immunopathology, mice with high-magnitude memory CD8 T cell responses were treated with neutralizing antibodies prior to RSV challenge. Neutralizing antibody treatment significantly reduced morbidity and prevented mortality following RSV challenge compared with IgG-treated controls. Neutralizing antibody treatment restricted early virus replication, which caused a substantial reduction in memory CD8 T cell activation and IFN-γ production, directly resulting in survival. In contrast, therapeutic neutralizing antibody administration did not impact morbidity, mortality, or IFN-γ levels, despite significantly reducing lung viral titers. Therefore, only pre-existing neutralizing antibodies prevent memory CD8 T cell-mediated immunopathology following RSV infection. Overall, our results have important implications for the development of future RSV vaccines.     

Pulmonary T cells induced by respiratory syncytial virus are functional and can make an important contribution to long-lived protective immunity

The contribution of T cell responses to immunity against respiratory syncytial virus (RSV) is not fully defined, but this is an important issue for vaccine design. Recent studies demonstrating RSV-induced pulmonary T cell suppression suggest that RSV may have evolved strategies to escape T cell immunity. Here we evaluated potential consequences of RSV-mediated immunosuppression for protective memory T cell responses in vivo. Surprisingly, we found strong ex vivo cytolytic activity and interferon production of pulmonary RSV-specific CD8(+) T cells both in the acute and the memory phase of primary murine RSV infection. More significantly, T cell memory made an important contribution to immunity against RSV independent of antibodies. Thus, RSV-primed mice were protected against challenge with RSV-recombinant vaccinia viruses, which can be controlled by RSV-specific T cells, but not by RSV-specific antibodies. In conclusion, RSV does not appear to impair acute and protective memory T cell responses induced by a primary infection. These findings further support that induction of T cell immunity should be a relevant goal for RSV vaccines.     

Antibody response and avidity of respiratory syncytial virus-specific total IgG, IgG1, and IgG3 in young children

Respiratory syncytial virus (RSV) is a major cause of acute respiratory disease in infants and young children. Considering that several aspects of the humoral immune response to RSV infection remain unclear, this study aimed to investigate the occurrence, levels, and avidity of total IgG, IgG1, and IgG3 antibodies against RSV in serum samples from children ≤5 years old. In addition, a possible association between antibody avidity and severity of illness was examined. The occurrence and levels of RSV-specific IgG depended on age, with infants <3 months old displaying high levels of antibodies, which were probably acquired from the mother. Children ≥24 months old also showed frequent occurrence and high levels of IgG, which was produced actively during infection. In addition, the avidity assay showed that the avidity of RSV-specific total IgG and IgG1 was lower in infants <3 months old who had acute respiratory disease than in age-matched controls. The avidity of RSV-specific IgG detected in children ≥24 months old with lower respiratory infection was lower than that in children with upper respiratory infection. These results indicate that the presence of high avidity RSV-specific IgG antibodies may lead to better protection against RSV infection in children <3 months old, who may have a lower probability of developing disease of increased severity. In addition, children ≥24 months old with RSV-specific IgG antibodies of low avidity tended to develop more severe RSV illness. These findings may be helpful in establishing vaccination schedules when a vaccine becomes available.     

Immunopathology of RSV infection: prospects for developing vaccines without this complication

Respiratory syncytial virus is the most important cause of lower respiratory tract infection in infants and young children. RSV clinical disease varies from rhinitis and otitis media to bronchiolitis and pneumonia. An increased incidence of asthma later in life has been associated with the more severe lower respiratory tract infections. Despite its importance as a pathogen, there is no licensed vaccine against RSV. This is due to a number of factors complicating the development of an effective and safe vaccine. The immunity to natural RSV infection is incomplete as re-infections occur in all age groups, which makes it challenging to design a protective vaccine. Second, the primary target population is the newborn infant, which has a relatively immature immune system and maternal antibodies that can interfere with vaccination. Finally, some vaccines have resulted in a predisposition for exacerbated pulmonary disease in infants, which was attributed to an imbalanced Th2-biased immune response, although the exact cause has not been elucidated. This makes it difficult to proceed with vaccine testing in infants. It is likely that an effective and safe vaccine needs to elicit a balanced immune response, including RSV-specific neutralising antibodies, CD8 T-cells, Th1/Th2 CD4 T-cells and preferably secretory IgA. Subunit vaccines formulated with appropriate adjuvants may be adequate for previously exposed individuals. However, intranasally delivered genetically engineered attenuated or vectored vaccines are currently most promising for newborns, as they are expected to induce a balanced immune response similar to that elicited to natural infection and not be subject to interference from maternal antibodies. Maternal vaccination may be the optimal strategy to protect the very young infants.     

Dissociation between serum neutralizing and glycoprotein antibody responses of infants and children who received inactivated respiratory syncytial virus vaccine.

The serum antibody response of infants and children immunized with Formalin-inactivated respiratory syncytial virus (RSV) vaccine 20 years ago was determined by using an enzyme-linked immunosorbent assay specific for the RSV fusion (F) and large (G) glycoproteins and a neutralization assay. Twenty-one young infants (2 to 6 months of age) developed a high titer of antibodies to the F glycoprotein but had a poor response to the G glycoprotein. Fifteen older individuals (7 to 40 months of age) developed titers of F and G antibodies comparable to those in children who were infected with RSV. However, both immunized infants and children developed a lower level of neutralizing antibodies than did individuals of comparable age with natural RSV infections. Thus, the treatment of RSV with Formalin appears to have altered the epitopes of the F or G glycoproteins or both that stimulate neutralizing antibodies, with the result that the immune response consisted largely of "nonfunctional" (i.e., nonneutralizing) antibodies. Subsequent natural infection of the vaccinees with wild-type RSV resulted in enhanced pulmonary disease. Despite this potentiation of illness, the infected vaccinees developed relatively poor G, F, and neutralizing antibody responses. Any or all of three factors may have contributed to the enhancement of disease in the RSV-infected vaccinees. First, nonfunctional antibodies induced by the inactivated RSV vaccine may have participated in a pulmonary Arthus reaction during RSV infection. Second, the poor antibody response of infants to the G glycoprotein present in the Formalin-inactivated vaccine may have been inadequate to provide effective resistance to subsequent wild-type virus infection. Third, the relatively reduced neutralizing antibody response of the infant vaccinees to wild-type RSV infection may have contributed to their enhanced disease by delaying the clearance of virus from their lungs.     

Infection of mice with respiratory syncytial virus during neonatal life primes for enhanced antibody and T cell responses on secondary challenge

Primary neonatal immune responses to infection or vaccines are weak when compared with those of adults. In addition, memory responses of neonatally primed animals may be absent, weak or T helper type 2 (Th2)-biased. Respiratory syncytial virus (RSV) is an important pathogen of human infants and infection during the neonatal period has been linked to the development of asthma in later life. Here we report that acute intranasal infection of neonatal mice with RSV induces significant RSV-specific antibody and CD8 T cell responses. These responses were boosted after RSV rechallenge during adulthood, demonstrating the establishment of memory after neonatal priming. Primary infection during neonatal life was associated, following rechallenge, with limited viral replication in the lung. Recall responses of both spleen and lymph node cells from neonatally primed and adult-primed mice were associated with interferon-gamma secretion, indicative of a Th1-type response. However, interleukin (IL)-4 and IL-5 secretion were enhanced only in spleen and lymph node cells from neonatally primed mice. Rechallenge of neonatally primed mice was also associated with increased concentrations of chemokines monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha and regulated upon activation normal T cell expressed and secreted in the lung. These may play a role in the enhanced inflammatory cell recruitment and immunopathology induced following RSV reinfection. Our results demonstrate therefore that immunity to RSV can be established during neonatal life and, importantly, that the quality of the subsequent response is dependent upon the age of first infection.     

Towards a human Lassa fever vaccine

Arenaviruses, such as Lassa fever, establish chronic infections in rodents, leading to incidental transmission to humans. Lassa fever is a clinically severe disease, yet the absence of second attacks implies life-long immunity. The aim of this review is to consider whether such immunity could be provided by vaccines. The South American arenaviruses are controlled by neutralising antibody and a clinical trial of live, attenuated vaccine for Argentinian haemorrhagic fever provided 84% protection. In contrast, there is no evidence for protective humoral immunity against Old World arenaviruses which are controlled by cell-mediated immune responses. Nevertheless, vaccination with Lassa glycoproteins can protect monkeys from disease, implying that protection may be achievable, even though the immunological mechanisms are distinct. Recombinant vaccinia viruses expressing various forms of Lassa glycoproteins can protect both guinea-pigs and primates, while additional protective responses can be mounted against nucleocapsid genes. However, vaccines based upon vaccinia constructs are no longer tenable for African populations with a high seroprevalence of HIV infection. The scientific challenge now remains to find alternative methods of delivering T-cell immunity against glycoproteins from Lassa virus in ways which can overcome the local economic and political hurdles to vaccine development.     

Intranasal immunisation with inactivated RSV and bacterial adjuvants induces mucosal protection and abrogates eosinophilia upon challenge

We have previously shown that following intranasal exposure to influenza virus, specific plasma cells are generated in the nasal-associated lymphoid tissue (NALT) and maintained for the life of the animal. However, we also showed that following infection with respiratory syncytial virus (RSV), specific plasma cells are generated in the NALT but wane quickly and are not maintained even after challenge, even though RSV-specific serum antibody responses remain robust. Only infection with influenza virus generated sterilising immunity, implying a role for these long-lived plasma cells in protection. We show here that the RSV-specific IgA NALT plasma cell population and lung antibody levels can be substantially boosted, both at acute and memory time points, by intranasal immunisation with inactivated RSV (iRSV) in combination with bacterial outer membrane vesicles (OMV) compared to live RSV alone. Finally, challenge with live RSV showed that immunisation with iRSV and OMV protect against both virus replication in the lung and the eosinophil infiltrate generated by either live RSV or iRSV alone. These data show that immunisation with iRSV and OMV maintains a NALT RSV-specific plasma cell population and generates an efficient protective immune response following RSV infection.     

Double-edged role of natural killer cells during RSV infection

Abstract

Natural killer cells play a vital role in the rejection of tumors and pathogen-infected cells. NK cells are indispensable in the early immune response against viral infections by directly targeting infected cells. Furthermore, NK cells influence adaptive immunity by driving virus-specific T-cell responses. Respiratory syncytial virus, a highly contagious virus that causes bronchiolitis, is the main reason for mortality in infants and elderly patients. RSV infection triggers both innate and adaptive immune responses. However, immunity against RSV is ephemeral due to the impaired development of immunological memory. The role of NK cells during RSV infection remains ambiguous. NK cells play a dual role in RSV infection; initially, their role is a protective one as they utilize their intrinsic cytotoxicity, followed by a detrimental one that induces lung injury due to the inhibition of antibody responses and the secretion of pro-inflammatory factors. Noteworthy, IFN-γ released from NK cells play a critical role in promoting a shift to adaptive responses and inhibiting antibody responses in neonates. Indeed, NK cells have a pro-inflammatory and inhibitory role rather than a cytotoxic one that contributes to the severity of the disease. Therapeutic options, including DNA-protein-based vaccines, synthetic peptides, and attenuated strains, are presently under tests. However, there is a need for effective strategies to augment NK cell activity and circumvent the pro-inflammatory activity to benefit the host. In this review, we focused on the role played by NK cells in the immune response and its outcome on the immunopathogenesis of RSV disease.     

Passively acquired antibodies to respiratory syncytial virus impair the secondary cytotoxic T-cell response in the neonatal mouse

Passively acquired antibody has been known since the 1940s to impair the secondary antibody response to the homologous antigen. However, the effect of passive immunity on the T-cell response is largely unknown. The results presented here demonstrate that monoclonal antibodies (Mabs) to respiratory syncytial virus (RSV), transferred in the mother's milk or injected directly, can reduce the generation of RSV-specific cytotoxic T-cell (Tc) precursors by the neonatal mouse; the development of influenza-specific Tc was unaffected. Both non-neutralizing and neutralizing antibodies, and Mabs directed against either the fusion (F) or G proteins of RSV, can impair the secondary Tc response. The ability of a given antibody to produce this impairment depends on its titre and its subclass, which determines its absorption from the gut by the neonate. These results are of interest in relation to virus infections in humans, such as RSV or measles, which are often contracted in the first 6 months of life, when maternal antibody is still present in high titre.     

The recall response induced by genital challenge with Chlamydia muridarum protects the oviduct from pathology but not from reinfection

The significant morbidities of ectopic pregnancy and infertility observed in women after Chlamydia trachomatis genital infection result from ascension of the bacteria from the endocervix to the oviduct, where an overly aggressive inflammatory response leads to chronic scarring and Fallopian tube obstruction. A vaccine to prevent chlamydia-induced disease is urgently needed. An important question for vaccine development is whether sterilizing immunity at the level of the oviduct is essential for protection because of the possibility that a chlamydial component drives a deleterious anamnestic T cell response upon oviduct reinfection. We show that mice inoculated with attenuated plasmid-cured strains of Chlamydia muridarum are protected from oviduct pathology upon challenge with wild-type C. muridarum Nigg despite induction of a response that did not prevent reinfection of the oviduct. Interestingly, repeated abbreviated infections with Nigg also elicited recall responses that protected the oviduct from pathology despite low-level reinfection of this vulnerable tissue site. Challenged mice displayed significant decreases in tissue infiltration of inflammatory leukocytes with marked reductions in frequencies of neutrophils but significant increases in frequencies of CD4 Th1 and CD8 T cells. An anamnestic antibody response was also detected. These data indicate that exposure to a live attenuated chlamydial vaccine or repeated abbreviated genital infection with virulent chlamydiae promotes anamnestic antibody and T cell responses that protect the oviduct from pathology despite a lack of sterilizing immunity at the site.     

Antibodies to the central conserved region of respiratory syncytial virus (RSV) G protein block RSV G protein CX3C-CX3CR1 binding and cross-neutralize RSV A and B strains

Respiratory syncytial virus (RSV) is a primary cause of severe lower respiratory tract disease in infants, young children, and the elderly worldwide, and despite decades of effort, there remains no safe and effective vaccine. RSV modifies the host immune response during infection by CX3C chemokine mimicry adversely affecting pulmonary leukocyte chemotaxis and CX3CR1+ RSV-specific T-cell responses. In this study we investigated whether immunization of mice with RSV G protein polypeptides from strain A2 could induce antibodies that block G protein-CX3CR1 interactions of both RSV A and B strains. The results show that mice immunized with RSV A2 G polypeptides generate antibodies that block binding of RSV A2 and B1 native G proteins to CX3CR1, and that these antibodies effectively cross-neutralize both A and B strains of RSV. These findings suggest that vaccines that induce RSV G protein-CX3CR1 blocking antibodies may provide a disease intervention strategy in the efforts to develop safe and efficacious RSV vaccines.     

Development of cell-mediated cytotoxic immunity to respiratory syncytial virus in human infants following naturally acquired infection

With virus-infected autologous and allogenic mononuclear cells as specific targets, the development of cell-mediated cytotoxic reactivity to respiratory syncytial virus (RSV) was studied in peripheral blood lymphocytes (PBL) in groups of infants with acute RSV infection and in other control groups of subjects during a community outbreak of RSV infection. No RSV-specific cellular cytotoxicity was observed in cord blood lymphocytes and in other uninfected controls. The PBL of infants with acute RSV infection exhibited significant cellular cytotoxic response. The activity peaked early, usually within 1 week after infection. The response appeared to be age-dependent. Over 65% of infants 6-24 months of age and about 35-38% of infants under 5 months of age exhibited cellular cytotoxicity to RSV. Cellular cytotoxic reactivity was observed against autologous and less frequently against allogenic RSV-infected target cells. These findings suggest the appearance of virus-specific cell-mediated cytotoxic immune response after acute RSV infection. The development of cellular cytotoxic responses may play a role in the mechanisms of protection against or the pathogenesis of RSV infection in man.     

Respiratory syncytial virus group-specific antibody response in nasopharyngeal secretions from infants and children after primary infection.

Respiratory syncytial virus (RSV) group-specific immunoglobulin A (IgA) and IgG enzyme-linked immunosorbent assay antibody and neutralizing antibody responses were determined for nasopharyngeal secretions (NPS) from 27 infants and children (6 to 18 months of age) undergoing primary infection with RSV group A or B strain. IgA and IgG antibody responses against RSV envelope glycoproteins (fusion [F] and large [G] glycoprotein) in NPS were also analyzed. Most subjects examined developed moderate levels of NPS IgA and IgG antibodies and neutralizing antibody activity to both group A and B strains in convalescent phase; however, the levels of antibodies to homologous strains were significantly higher than to the heterologous strains. Patients infected with group A developed antibodies in both F and G glycoproteins of A2 strains (group A). Patients infected with group B developed levels of antibody activity to F glycoprotein of A2 strain similar to those of patients infected with group A. However, these subjects developed little or no antibody response to G glycoprotein of A2 strain. These data suggest that the IgA and IgG antibody responses to G glycoprotein in the respiratory tract are group specific. It is suggested that lack of antibody response to the G glycoprotein of the heterologous group in the respiratory tract may determine the outcome of reinfection with other RSV strains.     

Formalin-inactivated respiratory syncytial virus vaccine induces antibodies to the fusion glycoprotein that are deficient in fusion-inhibiting activity.

The fusion (F) glycoprotein of respiratory syncytial virus (RSV) induces neutralizing antibodies and antibodies that inhibit fusion of infected cells (FI antibody). It was previously shown that infants and children immunized with Formalin-inactivated RSV 20 years ago developed antibodies that bound to the F glycoprotein but were deficient in neutralizing activity. A reexamination of these sera indicated that they were also deficient in FI activity. Thus, Formalin-inactivated RSV vaccine stimulated an unbalanced immune response in which an unusually large proportion of the induced antibodies were directed against nonprotective epitopes rather than against the epitopes that induce functional antibodies, i.e., neutralizing and FI antibodies. This deficiency in stimulation of functional antibodies probably decreased the protective efficacy of the vaccine and could have contributed to potentiation of disease in the vaccines during subsequent RSV infection.     

Antibody-dependent enhancement of respiratory syncytial virus infection by sera from young infants.

Respiratory syncytial virus (RSV) convalescent-phase sera and control sera from both infants ( < 6 months) and older individuals (1.5 to 90 years) were assayed for RSV-specific antibodies by neutralization, in vitro enhancing activity, and immunoprecipitation. Enhancement of RSV infection in U937 cells was demonstrated with convalescent-phase sera and was shown to be dependent on Fc receptors by blocking with human immunoglobulin G (P < 0.01). Convalescent-phase sera from infants enhanced infection at concentrations closer to physiological ones (10(-1) to 10(-3) dilutions of serum), while convalescent-phase sera from older individuals enhanced infection only at much lower concentrations (10(-3) to 10(-6) dilutions of serum; P < 0.01). To our knowledge, this is the first report of RSV-enhancing antibody activity in the sera of infants. The observed enhancing activity and the low neutralizing antibody levels are confined mostly to convalescent-phase sera from infants aged 0 to 6 months, suggesting that these factors may contribute to the increased severity of RSV disease frequently encountered in young infants.     

Human respiratory syncytial virus: pathogenesis,immune responses,and current vaccine approaches

Respiratory syncytial virus continues to pose a serious threat to the pediatric populations worldwide. With a genomic makeup of 15,200 nucleotides, the virus encodes for 11 proteins serving as envelope spikes, inner envelope proteins, and non-structural and ribonucleocapsid complexes. The fusion (F) and attachment (G) surface glycoproteins are the key targets for neutralizing antibodies. The highly variable G with altered glycosylations and the conformational alternations of F create challenges for vaccine development. The metastable F protein is responsible for RSV-host cell fusion and thus infectivity. Novel antigenic sites were identified on this form following its stabilization and solving its crystal structure. Importantly, site ø displays neutralizing activity exceeding those of post-F-specific and shared antigenic sites, such as site II which is the target for Palivizumab therapeutic antibody. Induction of high neutralizing antibody responses by pre-F immunization in animal models promoted it as a major vaccine candidate. Since RSV infection is more serious at age extremities and in individuals with undermining health conditions, vaccines are being developed to target these populations. Infants below three months of age have a suppressive immune system, making vaccines’ immunogenicity weak. Therefore, a suggested strategy to protect newborns from RSV infection would be through passive immunity of maternal antibodies. Hence, pregnant women at their third trimester have been selected as an ideal target for vaccination with RSV pre-F vaccine. This review summarizes the different modes of RSV pathogenesis and host’s immune response to the infection, and illustrates on the latest updates of vaccine development and vaccination approaches.     

Serum and nasal-wash immunoglobulin G and A antibody response of infants and children to respiratory syncytial virus F and G glycoproteins following primary infection.

An enzyme-linked immunosorbent assay (ELISA) with immunoaffinity-purified fusion (F) or attachment (G) glycoprotein was used to measure the serum and secretory immune responses of 18 infants and children, 4 to 21 months of age, who underwent primary infection with respiratory syncytial virus (RSV). Most of the 10 older individuals (9 to 21 months of age) developed moderate levels of serum and nasal-wash immunoglobin A (IgA) and IgG F and G antibodies. These individuals developed a moderate level of serum or nasal-wash antibodies that neutralized virus infectivity. One of the eight younger individuals (4 to 8 months of age) failed to develop an F antibody response, while three failed to develop a G antibody response. The most notable difference in the responses of the two age groups involved the titer in convalescent sera of G, F, and neutralizing antibodies which were 8- to 10-fold lower in younger individuals. Most of the younger infants failed to develop a rise in serum or nasal-wash neutralizing antibody. It is possible that the presence of maternally derived antibody in the younger infants suppressed the immune response to RSV infection, and that this accounted, in part, for the low level of postinfection antibody titer in this group. This low level and the irregular response of the infants less than 8 months of age may contribute to the severity of their initial infection and may also be responsible, in part, for their failure to develop effective resistance to subsequent reinfection by RSV.     

Identification of a novel human leucocyte antigen-A*01-restricted cytotoxic T-lymphocyte epitope in the respiratory syncytial virus fusion protein

Virus-specific cytotoxic T lymphocytes (CTL) play a major role in the clearance of respiratory syncytial virus (RSV) infection. To begin monitoring the immunological response to infection, especially in infants, it is important to identify human leucocyte antigen (HLA)-restricted CTL epitopes. Herein, we used a novel, comprehensive peptide panel containing all possible 8-, 9- and 10-mer peptides spanning the RSV fusion protein to screen for novel HLA-restricted T-cell epitopes. These peptide sets were synthesized as 10-mer peptides overlapping by nine amino acids and contained corresponding 8- and 9-mer peptides generated by C-terminal truncation. Unselected and uncultured peripheral blood mononuclear cells from healthy adult subjects were screened by interferon-gamma (IFN-gamma) Elispot assays against the peptide panel. Seven of 19 subjects displayed positive responses against 10 of the 565 peptides analysed. An HLA-A*01-restricted CTL epitope detected in three healthy adult subjects is characterized. This is the first RSV-specific memory CTL response identified in the fusion protein of RSV.     

Respiratory syncytial virus (RSV) vaccines--two steps back for one leap forward.

Respiratory viruses are among the most important causes of morbidity and mortality worldwide. From a vaccine viewpoint, such viruses may be divided into two principle groups-those where infection results in long-term immunity and whose continued survival requires constant mutation, and those where infection induces incomplete immunity and repeated infections are common, even with little or no mutation. Influenza virus and respiratory syncytial virus (RSV) typify the former and latter groups, respectively. Importantly, successful vaccines have been developed against influenza virus. However, this is not the case for RSV, despite many decades of research and several vaccine approaches. Similar to natural infection, the principle limitation of candidate RSV vaccines in humans is limited immunogenicity, characterised in part by short-term RSV-specific adaptive immunity. The specific reasons why natural RSV infection is insufficiently immunogenic in humans are unknown but circumvention of innate and adaptive immune responses are likely causes. Fundamental questions concerning RSV/host interactions remain to be addressed at both the innate and adaptive immune levels in humans in order to elucidate mechanisms of immune response circumvention. Taking the necessary steps back to generate such knowledge will provide the means to leap forward in our quest for a successful RSV vaccine. Recent developments relating to some of these questions are discussed.